Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 ( MCL1 ) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.

Acute lung injury (ALI), including its most severe form, acute respiratory distress syndrome (ARDS), is a common cause of acute hypoxemic respiratory failure. Although its clinical characteristics have been well characterized, the relevant mechanism remains unclear. An imbalance in autophagy leads to alveolar remodeling and triggers the pathogenesis of ARDS. In this study, we assessed the therapeutic efficacy of the STAT1 inhibitor fludarabine (Fluda) in ALI. C57BL6 mice were exposed to lipopolysaccharide (LPS), and their lung tissues were analyzed via next-generation transcriptome sequencing. Western blotting revealed that interferon regulatory factor 1 (IRF1) was highly expressed and STAT1 was phosphorylated following LPS exposure. Fluda significantly decreased the protein expression of STAT1/IRF1 and inhibited the alveolar infiltration of neutrophils and macrophages. Nitric oxide (NO), inducible nitric oxide synthase, tumor necrosis factor-α (TNF-α), interferon-γ, and interleukin-6 (IL-6) release was decreased in the lungs of mice and RAW264.7 macrophages following Fluda treatment. In LPS-induced GFP-LC3 transgenic mice treated with Fluda, the counts of LC3-expressing neutrophils and macrophages in bronchoalveolar (BAL) fluid were significantly decreased. Furthermore, Fluda decreased LC3 and p62 protein expression, thereby inhibiting the release of NO, IL-6, and TNF-α in BAL. In RAW264.7 cells, the inhibition of STAT1/IRF1 by Fluda decreased LPS-induced ERK and NF-κB p65 phosphorylation. The inhibition of STAT1/IRF1 by Fluda plays a pivotal role in modulating dysregulated autophagy by suppressing the MAPK and NF-κB p65 pathways in ALI.

Abstract BACKGROUND Pediatric high-grade gliomas (HGGs) present a therapeutic challenge due to their aggressive behavior and limited treatment options. Traditional treatments often fall short in addressing the genetic complexity and aggressiveness of pediatric HGGs, urging the search for and validation of novel genetic dependencies as potential therapeutic targets. METHODS Loss of function CRISPR screens enable the systematic investigation of gene function by selectively disrupting gene expression, offering invaluable insights into the genetic dependencies fuelling tumor growth and survival. Herein, we conducted genome-scale, loss of function CRISPR/Cas12 screens across genetically-defined subtypes of pediatric HGG, including diffuse midline gliomas (n=30 cell lines). RESULTS Distinct genetic dependencies exclusive to pediatric high-grade gliomas (pHGG) are uncovered compared to their adult counterparts, particularly within druggable pathways. These dependencies are associated with specific pathways such as cellular metabolism, epigenetics, and tissue development. Notably, histone-altered subtypes of pHGG exhibit dependency on distinct gene sets for growth. Furthermore, machine learning techniques are applied to correlate various -omics features (RNA, DNA, epigenomics, proteomics) with genetic dependencies, facilitating the identification of biomarkers predictive of therapeutic response. CONCLUSION Our work provides a comprehensive genetic dependency map of pHGG. We expect this data to serve as a springboard for enriching our biological understanding of pHGG, ultimately improving clinical outcomes and quality of life.

Pediatric high-grade gliomas (pHGGs) pose a significant challenge as the most aggressive central nervous system tumors in children. The lack of effective treatment and poor survival rates emphasize the critical need for innovative therapies to improve the prognosis of pediatric patients with pHGG. We executed CRISPR-cas9 knockout (KO) screenings in 65 pediatric and 10 adult high-grade glioma (HGG) cell lines to explore unique functional dependencies associated with pHGGs. Drug assays were carried out to assess the targetability of a gene dependency of interest in pHGG cell lines. Subsequently, Random Forest machine learning algorithm was employed on ‘omics’ datasets to identify biomarkers indicative of drug response. Finally, biomarkers were confirmed through sequencing-based methods across various pediatric cancers. CRISPR-cas9 KO screens revealed 8 crucial genes essential for pHGG growth, namely MCL-1, ATIC, DHFR, EED, HDAC2, PARP1, PDK1, PIK3CA and TYMS. Among these, Myeloid Cell Leukemia (MCL-1) was further characterized as it emerged as the top differential genetic dependency in pHGGs. Consistent with this, MCL1 inhibitors exhibited potent anti-cancer activity on 43% of pHGG cell lines. Multi-feature prediction analysis identified 140 features correlated to response. Strikingly, a previously undescribed methylation site within the BCL2L1 locus, cg00300298, ranked in the top 5% of features. Tissue analysis revealed a wide range of other pediatric brain cancers that harbor the BCL2L1 cg00300298 methylation mark as compared to non-malignant brain tissue. Lastly, we validated the utility of BCL2L1 methylation as a predictive biomarker of MCL1 inhibitor sensitivity across a large panel of pediatric brain cancer cell lines. Using an integrated genomic approach, we identify MCL1 as a distinct therapeutic target in BCL2L1-methylated pediatric brain cancers. This offers a rational approach for stratifying patients who may benefit from MCL1 inhibitors.

To achieve high performance in esports, players must be able to effectively and efficiently control input devices such as a computer mouse and keyboard (i.e., input skills). Characterizing and quantifying a player’s input skills can provide useful insights, but collecting and analyzing sufficient amounts of data in ecologically valid settings remains a challenge. Targeting the popular esports game, League of Legends, we go beyond the limitations of previous studies and demonstrate a holistic pipeline of input behavior analysis: from quantifying the quality of players’ input behavior (i.e., input skill) to training players based on the analysis. Based on interviews with five top-tier professionals and analysis of input behavior logs from 4,835 matches played freely at home collected from 193 players (including 18 professionals), we confirmed that players with higher ranks in the game implement eight different input skills with higher quality. In a three-week follow-up study using a training aid that visualizes a player’s input skill levels, we found that the analysis provided players with actionable lessons, potentially leading to meaningful changes in their input behavior.

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1-0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

The invisibility of a physical outline to constrain users within an area for conducting their gesture commands frequently results in making errors, which may further affect the less use of the gesture inputs. In this, despite the advantages of gestural user interface (UI), those were rarely used in our home electronics. This paper explores the possibilities of sensing and constraining the gesture interaction through the design, development, and user study of a research prototype called Hand-in-O. It is a device that constrains users in performing gestures by moving the hands inside of the frame to provide light and sound feedback. Using this, we conducted use experience exploration and a design workshop upon twenty-one participants. From the study, we could obtain categories for suggested usages of Hand-in-O in household contexts. Furthermore, using the frame form’s affordance, we found it may attract users’ curiosity about performing gesture interactions to control various media in homes.

Formyl peptide receptors (FPRs) are mainly expressed on leucocytes and sense microbe-associated molecular pattern (MAMP) molecules, thereby regulating leukocyte chemotaxis and activation. The formyl peptide receptor 2 (FPR2) selective agonist WKYMVm (Trp-Lys-Met-Val-D-Met) has shown potent pro-angiogenic, anti-inflammatory, and anti-apoptotic properties. In this study, we investigated whether WKYMVm exhibits bactericidal activity during neutrophil accumulation in acute lung injury (ALI) in mice and determined its cellular signaling pathways in HL-60 neutrophil-like cells. A daily intraperitoneal treatment of ALI mice with WKYMVm (2.5- and 5 mg/kg/d) daily over four days decreased the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β, while it increased the MPO and NO release by differentiated HL-60 neutrophil-like cells. The IRF1 level and STAT1 phosphorylation at S727 were increased in the lungs of mice with ALI treated with WKYMVm. Lung histology induced by ALI was unaffected by treatment with WKYMVm. In vitro, WKYMVm increased MPO, NO, and SOD activity, as well as IRF1 and STAT1 phosphorylation at Ser727. Taken together, our data suggest therapeutic potential of WKYMVm, via FPR2-dependent regulation of STAT1/IRF1, in ALI.

High-throughput nucleotide sequencing is promising for respiratory infections diagnostic.However, determining the causative pathogen from colonization with analysis of massive bacterial 16s rRNA gene sequencing is challenging.Instead of removing human reads, targeting both human and bacterial gene amplicons into next-generation sequencing (NGS) can be utilized for indexing pathogen.This study aimed to investigate human and bacterial reads as an index for detecting pathogens in respiratory tract infections.Sputum and bronchoalveolar lavage fluid (BALF) were obtained from patients hospitalized at Jichi Medical University Hospital, Tochigi, Japan.The target amplicons were human HSD11B2 gene and 16s rRNA genes from 52 common human pathogens.Multiplex PCR with adaptor and sample identifier was appended prior to run on MiSeq.Ruby program was customized to analyze the reads against the local database based on Blast 2.8.1+ algorithm reporting best-matched sequences and bit score for sequence similarity.Eighty-five samples reported 83330 top hits queries with median bit score was 836 .The reads distribution between sputum (80388) and BALF (2942) were different (P <0.05) consist of H. sapiens (29%;80%), S. pneumoniae (18%;7%), H. parainfluenza (11%;0.5%),N. gonorrhoeaea (11%;0.9%),and P. aeruginosa (7%;0.1%).Significant differences of pathogen reads (606;5) and pathogen-human ratio (8.13;0.62)were found between sputum and BALF.This study suggests that targeting human and bacterial genes providing balanced analysis for pathogen index may hold promise as new microbiological techniques.

High-throughput nucleotide sequencing is promising for respiratory infections diagnostic.However, determining the causative pathogen from colonization with analysis of massive bacterial 16s rRNA gene sequencing is challenging.Instead of removing human reads, targeting both human and bacterial gene amplicons into next-generation sequencing (NGS) can be utilized for indexing pathogen.This study aimed to investigate human and bacterial reads as an index for detecting pathogens in respiratory tract infections.Sputum and bronchoalveolar lavage fluid (BALF) were obtained from patients hospitalized at Jichi Medical University Hospital, Tochigi, Japan.The target amplicons were human HSD11B2 gene and 16s rRNA genes from 52 common human pathogens.Multiplex PCR with adaptor and sample identifier was appended prior to run on MiSeq.Ruby program was customized to analyze the reads against the local database based on Blast 2.8.1+ algorithm reporting best-matched sequences and bit score for sequence similarity.Eighty-five samples reported 83330 top hits queries with median bit score was 836 .The reads distribution between sputum (80388) and BALF (2942) were different (P <0.05) consist of H. sapiens (29%;80%), S. pneumoniae (18%;7%), H. parainfluenza (11%;0.5%),N. gonorrhoeaea (11%;0.9%),and P. aeruginosa (7%;0.1%).Significant differences of pathogen reads (606;5) and pathogen-human ratio (8.13;0.62)were found between sputum and BALF.This study suggests that targeting human and bacterial genes providing balanced analysis for pathogen index may hold promise as new microbiological techniques.

Background and Objectives Treatment with mesenchymal stem cells (MSC) in spinal cord injury (SCI) has been highlighted as therapeutic candidate for SCI. Although astrogliosis is a major phenomenon after SCI, the role of astrogliosis is still controversial. In this study, we determined whether acute transplantation of MSC improves the outcome of SCI through modulating astrogliosis. Methods Bone marrow derived rat MSCs were induced neural differentiation and transplanted after acute SCI rats. Matrix metalloproteinase (MMP) and neuro-inflammatory pathway were analyzed for acute astrogliosis at 1, 3 and 7 d after SCI in RT-PCR- and western blot analysis. Functional outcome was assessed serially at postoperative 1 d and weekly for 4 weeks. Histopathologic analysis was undertaken at 7 and 28 d following injury in immunohistochemistry. Results Transplantation of MSCs decreased IL-1α, CXCL-2, CXCL-10, TNF-α and TGF-β in a rat model of contusive SCI. Protein level of NF-κB p65 was slightly decreased while level of STAT-3 was increased. In immunohistochemistry, MSC transplantation increased acute astrogliosis whereas attenuated scar formation with increased sparing white matter of spinal cord lesions. In RT-PCR analysis, mRNA levels of MMP2 was significantly increased in MSC transplanted rats. In BBB locomotor scale, the rats of MSC treated group exhibited improvement of functional recovery. Conclusions Transplantation of MSC reduces the inflammatory reaction and modulates astrogliosis via MMP2/STAT3 pathway leading to improve functional recovery after SCI in rats.

Background: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. Methods: Mouse AE II cell line MLE-12 were exposed to 1-10 mu g/mL BLM and 0.01-100 mu M baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. Results: BLM decreased the proliferation of MLE 12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor alpha, and transforming growth factor beta 1. Based on Hoechst 33258 staining BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. Conclusion: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

WITHDRAWN AO1810 SMALL MOLECULE A ATTENUATES LIPOPOLYSACCHARIDES INDUCED-INOS AND INFLAMMATION IN ALI MICE MODEL JOOYEON LEE, HANBYEOL LEE, YOUNGHEON PARK, SE-RAN YANG Department of Thoracic and Cardiovascular Surgery, School of Medicine, Kangwon National University, Chuncheon, Gangwon Republic of Korea Background and Aims: Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS), are common causes of acute hypoxemic respiratory failure in the critically ill patient and characterized by lung oedema, endothelial and epithelial injury and immune cell infiltration. Although antibiotic therapy and intensive care are developing, the therapeutic mechanism of ALI remains poor. In this study, we investigated which pathway is associated with ALI pathology and developed the therapeutic approach using small molecule A. Methods: Lipopolysaccharides (LPS) is well known as lipoglycans and endotoxins, which is found in the outer membrane of Gram-negative bacteria. We established ALI animal model using LPS that is injected to mice for 24h intratracheally. To determine therapeutic effect of small molecule A, we pre-treated small molecule A intratracheally before injection of LPS. In vivo, we investigated histological changes in H&E staining, related pathway in western blotting and gene mRNA levels. Results: We found that small molecule A decreased LPS-induced secretion of TNF-α, IL6, NO and expression of iNOS in mice. Moreover, we determined that small molecule A also attenuated LPS-induced NO and inflammatory cytokines, which are accompanied with suppressing of NF-kB p65 signalling in RAW 264.7. Conclusion: Taken together, this study for the first time unveils that small molecule A inhibits LPS-induced ALI in vitro and in vivo, and it might be used potential candidates for trials of early treatment of ALI. Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF2017R1A2B4006197, NRF-2017M3A9B4051542).

sitivity pneumonitis induced by cryptococcus neoformans. Results: Mr. Wong, an 81-year-old male, who used to sell herbal tea for living, suffered from chest pain for one month. He also complained progressed dyspnea in recently one week. He denied fever, legs pitting edema, productive cough, and contact or travel history. He visited our clinics on Feb. 6, 2018. His saturation was around 80% in ambient air. Therefore he was admitted for survey. His medical history included asthma, coronary artery disease, hypertension, old pulmonary tuberculosis, gout and old cerebral vascular accident. His current medicine was betaloc, aspirin, MgO, sevikar, tonsaric, vytorin, and acetylcystein. Chest CT showed bilateral ground grass opacity from upper to lower lobe, and fibrotic change over lower lobe bilaterally. His white blood cell counts was 10,200/cumm and CRP 11 mg/dl. Empiric antibiotics tazocin was given. There were negative results including serum mycoplasma IgM, Cryptococcus neoformans antigen, blood culture and sputum culture. DLCO was only 25%. Due to history of coronary artery disease and chest pain persisted, cardiac catheterization was performed and stenting from left anterior descending and left circumflex artery. But his chest pain persisted with persisted low saturation. Methylprednisolone intravenous use was given for suspect hypersensitivity pneumonitis. Therefore we arranged bronchoscopic examination. Bronchoalveolar lavage via RB2 was performed. Fluid analysis showed lymphocytosis (37%). The fungus culture showed cryptococcus neoformans (100 CFU/mL). According to the results, diflucan intravenous was treated. His symptoms improved and saturation was up to 95% without oxygen use. Conclusion: Exposure to fungus antigen should be keep in mind in clinical evaluation of susceptible patients.

The chronic obstructive pulmonary disease (COPD) is one of the common chronic inflammatory disease following cough, phlegm and breathing disorder. Recently, prevalence rate and mortality of COPD are much higher with high smoking rate, therefore the therapeutic approach are emerging for COPD. The cigarette smoke (CS)-induced alveolar epithelial injury and energy metabolic disorders are known as cause of pathophysiologic changes with COPD. Indeed, CS also increase inflammatory response, reactive oxygen species (ROS) and nitric oxide (NO) and activates NF-κB/MAPKs pathway in alveoli. Interestingly, we found a small molecules that alleviate CS-induced alveolar injury by improving cellular metabolism in alveolar epithelial cells. To demonstrate the effect of small molecule C, we induced experimental COPD using cigarette smoke extract. The murine alveolar epithelial cells (MLE-12) were exposed by 0.6% cigarette smoke extract (CSE) for 24 h, and 0.1-3 μM small molecule C was pre-treated for 24 h before the exposure with CSE. The small molecule C enhances cell proliferation with anti-apoptosis and reduces inflammatory cytokines, tumor necrosis factor-α and interleukin-6, intracellular ROS/NO and NF-κB/MAPKs pathway activity. Indeed, we demonstrated that the small molecule C also activates c-AMP/CREB/PKA pathway signaling by western blotting. Therefore, these findings suggest that the small molecule has therapeutic effect through cellular metabolic improvement, anti-inflammatory and -apoptotic functions in COPD. Acknowledgement this work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2017M3A9B4051542).

The oxidative stress and cellular apoptosis by environmental factor including cigarette smoke induces alveolar airway remodeling leading to chronic obstructive pulmonary disease (COPD). Recently, the receptor for advanced glycan end products (RAGE) which is highly expressed in alveolar epithelium is emerging as a biomarker for COPD susceptibility or progression. However, it still remains unknown how RAGE plays a role in cigarette smoke extract (CSE)-exposed human alveolar type II epithelial cell line. Therefore, we determined the efficacy of RAGE-specific antagonist FPS-ZM1 in response to CSE-induced lung epithelial cells. CSE induced the elevated generation of RONS and release of pro-inflammatory cytokines, and impaired the cellular antioxidant defense system. Further, CSE induced the alteration of RAGE distribution via the activation of redox-sensitive DAMP (Damage-associated molecular patterns) signaling through Nrf2 in cells. Although pre-treatment with SB202190 (p38 inhibitor) or SP600125 (JNK inhibitor) failed to recover the alteration of RAGE distribution, treatment of FPS-ZM1 significantly exhibited anti-inflammatory and anti-oxidative/nitrosative effects, also inhibited the activation of redox-sensitive DAMP signaling through Nrf2 (nuclear factor erythroid 2-related factor 2) migration in the presence of CSE. Taken together, our data demonstrate that RAGE and Nrf2 play a pivotal role in maintenance of alveolar epithelial integrity.