Targeted delivery of HSP90 inhibitors for efficient therapy of CD44-positive acute myeloid leukemia and solid tumor-colon cancer.

Heat shock protein 90 (HSP90) is overexpressed in numerous cancers, promotes the maturation of numerous oncoproteins and facilitates cancer cell growth. Certain HSP90 inhibitors have entered clinical trials. Although less than satisfactory clinical effects or insurmountable toxicity have compelled these trials to be terminated or postponed, these results of preclinical and clinical studies demonstrated that the prospects of targeting therapeutic strategies involving HSP90 inhibitors deserve enough attention. Nanoparticulate-based drug delivery systems have been generally supposed as one of the most promising formulations especially for targeting strategies. However, so far, no active targeting nano-formulations have succeeded in clinical translation, mainly due to complicated preparation, complex formulations leading to difficult industrialization, incomplete biocompatibility or nontoxicity. In this study, HSP90 and CD44-targeted A6 peptide functionalized biomimetic nanoparticles (A6-NP) was designed and various degrees of A6-modification on nanoparticles were fabricated to evaluate targeting ability and anticancer efficiency. With no excipients, the hydrophobic HSP90 inhibitor G2111 and A6-conjugated human serum albumin could self-assemble into nanoparticles with a uniform particle size of approximately 200 nm, easy fabrication, well biocompatibility and avoidance of hepatotoxicity. Besides, G2111 encapsulated in A6-NP was only released less than 5% in 12 h, which may avoid off-target cell toxicity before entering into cancer cells. A6 peptide modification could significantly enhance uptake within a short time. Moreover, A6-NP continues to exert the broad anticancer spectrum of Hsp90 inhibitors and displays remarkable targeting ability and anticancer efficacy both in hematological malignancies and solid tumors (with colon tumors as the model cancer) both in vitro and in vivo. Overall, A6-NP, as a simple, biomimetic and active dual-targeting (CD44 and HSP90) nanomedicine, displays high potential for clinical translation.

Unlocking the Potential: A Systematic Review of Master Protocol in Pediatrics.

The use of master protocols allows for innovative approaches to clinical trial designs, potentially enabling new approaches to operations and analytics and creating value for patients and drug developers. Pediatric research has been conducted for many decades, but the use of novel designs such as master protocols in pediatric research is not well understood. This study aims to provide a systematic review on the utilization of master protocols in pediatric drug development. A search was performed in September 2022 using two data sources (PubMed and ClinicalTrials.gov) and included studies conducted in the past10 years. General study information was extracted such as study type, study status, therapeutic area, and clinical trial phase. Study characteristics that are specific to pediatric studies (such as age of the participants and pediatric drug dosing) and important study design elements (such as number of test drug arms and whether randomization and/or concurrent control was used) were also collected. Our results suggest that master protocol studies are being used in pediatrics, with platform and basket trials more common than umbrella trials. Most of this experience is in oncology and early phase studies. There is a rise in the use starting in 2020, largely in oncology and COVID-19 trials. However, adoption of master protocols in pediatric clinical research is still on a small scale and could be substantially expanded. Work is required to further understand the barriers in implementing pediatric master protocols, from setting up infrastructure to interpreting study findings.

Efficacy and Safety of BTKis in Central Nervous System Lymphoma: A Systematic Review and Meta-Analysis.

BACKGROUND: This systematic review and meta-analysis aimed to evaluate the efficacy and safety of Bruton tyrosine kinase inhibitors (BTKis) for central nervous system lymphoma (CNSL). METHODS: A systematic review was carried out to identify relevant studies from the PubMed, Embase, Cochrane Library, Web of Science, WanFang, CNKI, and CBM databases. The studies included patients with CNSL who received BTKis and reported the overall response (OR), complete remission (CR), and partial response (PR). An overall effect analysis was performed using STATA 15.0. A random-effects model was utilized to calculate the pooled rates, and 95% confidence intervals (CI) were determined for all outcomes. RESULTS: A total of 21 studies involving 368 patients were included in the meta-analysis. For newly diagnosed CNSL, due to the small simple size, we conducted a quantitative description, and the ORR could reach up to 100%. For relapsed/refractory patients, the pooled ORR was 72% (95% CI: 64-80%, I2 = 54.89%, p = 0.00), with a pooled CR and PR of 43% (95% CI: 33-54%, I2 = 65.40%, p = 0.00) and 23% (95% CI: 13-35%, I2 = 78.05%, p = 0.00), respectively. Most adverse events were hematology-related and generally manageable. CONCLUSION: BTKis showed acceptable efficacy and safety in treating patients with CNSL. However, large and well-designed trials are still required to confirm BTKis as a treatment for CNSL.

[Review and prospect of traditional Chinese medicine in treatment of trauma based on bibliometrics].

This study aims to summarize the research hotspots and trends in traditional Chinese medicine(TCM) treatment of trauma and provide suggestions for collaborative research on the treatment of trauma with integrated Chinese and western medicine. The re-levant research articles on TCM treatment of trauma were searched against CNKI, Wanfang, VIP, Web of Science, and PubMed from inception to December 31, 2022 and analyzed using bibliometric. After screening, 315 articles in Chinese and 34 articles in English were included. The articles were mainly published by TCM journals. The core author groups were absent. The author affiliations were dominated by medical institutions and supplemented by universities, research institutions, and government agencies. The research mainly focused on the clinical practice and trials of TCM in trauma treatment. Although the TCM treatment of trauma emerged early, it is still in the initial stage of development. The future research can focus on innovating the service model of trauma treatment, improving clinical research capability, improving medical quality management, and strengthening the TCM talent teams in comprehensive hospitals. Western medicine is precise and effective in the treatment of trauma, while TCM with unique treatment methods and effects can play a complementary role. Therefore, the trauma treatment with integrated Chinese and western medicine demonstrates a promising prospect.

Short-stranded DNA segment-modulated LAMP/H+ as signal transducer to guide CHA-cooperated amplifiable electrochemical biosensing.

BACKGROUND: Modulating loop-mediated isothermal amplification (mLAMP) by short-stranded DNA segment trigger (T) to generate byproducts H+ ions (mLAMP/H+) as signal transducer is intriguing for developing catalytic hairpin assembly (CHA)-cooperated amplifiable electrochemical biosensors. This would be a big challenge for traditional LAMP that is basically suitable for amplifying long-stranded oligonucleotides up to 200-300 nt. To address this inherent limitation of traditional LAMP, many researchers have put in efforts to explore improvements in this that would allow LAMP to be used for a wider range of target species amplification. RESULTS: Here in this work, we are inspired to explore two-step loop-mediated amplification, firstly forming T-activated double-loop dumbbell structure (DLDS) intermediate by a recognition hairpin and a hairpin precursor, and next DLDS-guided mLAMP process with the aid of two primers to yield mLAMP/H+ during successive DNA incorporation via nucleophilic attacking interaction. To manipulate the mLAMP/H+-directed transduction of input T, a pH-responsive triplex strand is designed with the ability of self-folding in Hoogsteen structure at slightly acidic conditions, resulting in the dehybridization of a fuel strand (FS) to participate in CHA between two hairpins on the modified electrode surface, in which FS is repetitively displaced and recycled to fuel the progressive CHA events. In the as-assembled dsDNA complexes, numerous electroactive ferrocene labels are immobilized in the electrode sensing interface, thereby generating significantly amplified electrochemical current signal that can sense the presented and varied T. SIGNIFICANCE: It is clear that we have creatively constructed a unique electrochemical biosensor for disease detection. Benefited from the rational combination of mLAMP and CHA, our electrochemical strategy is highly sensitive, specific and simplified, and would provide a new paradigm to construct various mLAMP/H+-based biosensors for other short-stranded DNA or microRNAs markers.

A Patient Similarity Network (CHDmap) to Predict Outcomes After Congenital Heart Surgery: Development and Validation Study.

Background: Although evidence-based medicine proposes personalized care that considers the best evidence, it still fails to address personal treatment in many real clinical scenarios where the complexity of the situation makes none of the available evidence applicable. "Medicine-based evidence" (MBE), in which big data and machine learning techniques are embraced to derive treatment responses from appropriately matched patients in real-world clinical practice, was proposed. However, many challenges remain in translating this conceptual framework into practice. Objective: This study aimed to technically translate the MBE conceptual framework into practice and evaluate its performance in providing general decision support services for outcomes after congenital heart disease (CHD) surgery. Methods: Data from 4774 CHD surgeries were collected. A total of 66 indicators and all diagnoses were extracted from each echocardiographic report using natural language processing technology. Combined with some basic clinical and surgical information, the distances between each patient were measured by a series of calculation formulas. Inspired by structure-mapping theory, the fusion of distances between different dimensions can be modulated by clinical experts. In addition to supporting direct analogical reasoning, a machine learning model can be constructed based on similar patients to provide personalized prediction. A user-operable patient similarity network (PSN) of CHD called CHDmap was proposed and developed to provide general decision support services based on the MBE approach. Results: Using 256 CHD cases, CHDmap was evaluated on 2 different types of postoperative prognostic prediction tasks: a binary classification task to predict postoperative complications and a multiple classification task to predict mechanical ventilation duration. A simple poll of the k-most similar patients provided by the PSN can achieve better prediction results than the average performance of 3 clinicians. Constructing logistic regression models for prediction using similar patients obtained from the PSN can further improve the performance of the 2 tasks (best area under the receiver operating characteristic curve=0.810 and 0.926, respectively). With the support of CHDmap, clinicians substantially improved their predictive capabilities. Conclusions: Without individual optimization, CHDmap demonstrates competitive performance compared to clinical experts. In addition, CHDmap has the advantage of enabling clinicians to use their superior cognitive abilities in conjunction with it to make decisions that are sometimes even superior to those made using artificial intelligence models. The MBE approach can be embraced in clinical practice, and its full potential can be realized.

Considerations for master protocols using external controls.

There has been an increasing use of master protocols in oncology clinical trials because of its efficiency to accelerate cancer drug development and flexibility to accommodate multiple substudies. Depending on the study objective and design, a master protocol trial can be a basket trial, an umbrella trial, a platform trial, or any other form of trials in which multiple investigational products and/or subpopulations are studied under a single protocol. Master protocols can use external data and evidence (e.g. external controls) for treatment effect estimation, which can further improve efficiency of master protocol trials. This paper provides an overview of different types of external controls and their unique features when used in master protocols. Some key considerations in master protocols with external controls are discussed including construction of estimands, assessment of fit-for-use real-world data, and considerations for different types of master protocols. Similarities and differences between regular randomized controlled trials and master protocols when using external controls are discussed. A targeted learning-based causal roadmap is presented which constitutes three key steps: (1) define a target statistical estimand that aligns with the causal estimand for the study objective, (2) use an efficient estimator to estimate the target statistical estimand and its uncertainty, and (3) evaluate the impact of causal assumptions on the study conclusion by performing sensitivity analyses. Two illustrative examples for master protocols using external controls are discussed for their merits and possible improvement in causal effect estimation.

ZCHSound: Open-Source ZJU Paediatric Heart Sound Database With Congenital Heart Disease.

BACKGROUND: Congenital heart disease (CHD) is a common birth defect in children. Intelligent auscultation algorithms have been proven to reduce the subjectivity of diagnoses and alleviate the workload of doctors. However, the development of this algorithm has been limited by the lack of reliable, standardized, and publicly available pediatric heart sound databases. Therefore, the objective of this research is to develop a large-scale, high-standard, high-quality, and accurately labeled pediatric congenital heart disease (CHD) heart sound database, and perform classification tasks to evaluate its performance, filling this important research gap. METHOD: From 2020 to 2022, we collaborated with experienced cardiac surgeons from Zhejiang University Children's Hospital to collect heart sound signals from 1259 participants using electronic stethoscopes. To ensure accurate disease diagnosis, the cardiac ultrasound images for each participant were acquired by an experienced ultrasonographer, and the final diagnosis was confirmed through the consensus of two cardiac experts or cardiac surgeons. To establish the benchmark of ZCHSound, we extracted 84 time-frequency features from the heart sounds and evaluated the performance of the classification task using machine learning models. Additionally, we evaluated the importance scores of the 84 features in distinguishing between normal and pathological heart sounds in children using SHapley Additive exPlanations (SHAP) values. RESULTS: The ZCHSound database contains heart sound data from 1259 participants, with all data divided into two datasets: one is a high-quality, filtered clean heart sound dataset, and the other is a low-quality, noisy heart sound dataset. In the evaluation of the high-quality dataset, our random forest ensemble model achieved an F1 score of 90.3% in the classification task of normal and pathological heart sounds. Moreover, the SHAP analysis results demonstrate that frequency-domain features have a more significant impact on the model output compared to time-domain features. Features related to the cardiac diastolic period have a greater influence on the model's classification results compared to those related to the systolic period. CONCLUSION: This study has successfully established a large-scale, high-quality, rigorously standardized pediatric CHD sound database with precise disease diagnosis. This database not only provides important learning resources for clinical doctors in auscultation knowledge but also offers valuable data support for algorithm engineers in developing intelligent auscultation algorithms. Our data can be accessed and downloaded by the public at http://zchsound.ncrcch.org.cn/.

[Knockdown of interferon-γ inducible protein 30 (IFI30) inhibits the proliferation, invasion and migration of human glioma U251 cells by activating STAT1 and promotes their apoptosis].

Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the TranswellTM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.

A developmental critical period for ocular dominance plasticity of binocular neurons in mouse superior colliculus.

Detecting visual features in the environment is crucial for animals' survival. The superior colliculus (SC) is implicated in motion detection and processing, whereas how the SC integrates visual inputs from the two eyes remains unclear. Using in vivo electrophysiology, we show that mouse SC contains many binocular neurons that display robust ocular dominance (OD) plasticity in a critical period during early development, which is similar to, but not dependent on, the primary visual cortex. NR2A- and NR2B-containing N-methyl-D-aspartate (NMDA) receptors play an essential role in the regulation of SC plasticity. Blocking NMDA receptors can largely prevent the impairment of predatory hunting caused by monocular deprivation, indicating that maintaining the binocularity of SC neurons is required for efficient hunting behavior. Together, our studies reveal the existence and function of OD plasticity in SC, which broadens our understanding of the development of subcortical visual circuitry relating to motion detection and predatory hunting.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

BACKGROUND: Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. RESULTS: First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. CONCLUSION: Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Tumor-Agnostic Approvals: Insights and Practical Considerations.

Since the first approval of a tumor-agnostic indication in 2017, a total of seven tumor-agnostic indications involving six drugs have received approval from the FDA. In this paper, the master protocol subteam of the Statistical Methods in Oncology Scientific Working Group, Biopharmaceutical Session, American Statistical Association, provides a comprehensive summary of these seven tumor-agnostic approvals, describing their mechanisms of action; biomarker prevalence; study design; companion diagnostics; regulatory aspects, including comparisons of global regulatory requirements; and health technology assessment approval. Also discussed are practical considerations relating to the regulatory approval of tumor-agnostic indications, specifically (i) recommendations for the design stage to mitigate the risk that exceptions may occur if a treatment is initially hypothesized to be effective for all tumor types and (ii) because drug development continues after approval of a tumor-agnostic indication, recommendations for further development of tumor-specific indications in first-line patients in the setting of a randomized confirmatory basket trial, acknowledging the challenges in this area. These recommendations and practical considerations may provide insights for the future development of drugs for tumor-agnostic indications.

An Overview of Current Statistical Methods for Implementing Quality Tolerance Limits.

BACKGROUND: In 2016, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use updated its efficacy guideline for good clinical practice and introduced predefined quality tolerance limits (QTLs) as a quality control in clinical trials. QTLs are complementary to Quality by Design (QbD) principles (ICH-E8) and are one of the components of the risk-based clinical trial quality management system. METHODS: Currently the framework for QTLs process is well established, extensively describing the operational aspects of Defining, Monitoring and Reporting, but a single source of commonly used methods to establish QTLs and secondary limits is lacking. This paper will primarily focus on closing this gap and include applications of statistical process control and Bayesian methods on commonly used study level quality parameters such as premature treatment discontinuation, study discontinuation and significant protocol deviations as examples. CONCLUSIONS: Application of quality tolerance limits to parameters that correspond to critical to quality factors help identify systematic errors. Some situations pose special challenges to implementing QTLs and not all methods are optimal in every scenario. Early warning signals, in addition to QTL, are necessary to trigger actions to further minimize the possibility of an end-of-study excursion.

Evaluation of bioequivalence and safety analysis of capecitabine tablets and Xeloda® under postprandial dosing conditions in Chinese patients with solid tumor.

OBJECTIVES: To compare the pharmacokinetic and safety of the test group capecitabine tablets (0.5 g) and the reference group capecitabine tablets (0.5 g). METHODS: This study was registered at www.chinadrugtrials.org.cn under the registration number CTR20220138. 48 subjects with solid tumor were recruited and randomized to receive either the test group or the reference group at a dose of 2 g per cycle for three cycles of the entire trial. RESULTS: The point estimate of the geometric mean ratio of Cmax for the subject and reference groups was 1.0670, which was in the range of 80.00%-125.00%. And the upper limit of 95% confidence interval was -0.0450 < 0. The statistics of geometric mean ratio of AUC0-t and AUC0-∞ (test group/reference group) and their 90% confidence intervals were in the range of 80.00%-125.00%, thus the test group was bioequivalent to the reference group under the conditions of this postprandial test. There were no major or serious adverse events. Conclusion: The pharmacokinetic profiles of capecitabine under postprandial conditions were consistent between the two groups. The two groups were bioequivalent and had a similar favorable safety profile in Chinese patients with solid tumor.

Cerebellar contributions to fear-based emotional processing: relevance to understanding the neural circuits involved in autism.

Cerebellar networks have traditionally been linked to sensorimotor control. However, a large body of evidence suggests that cerebellar functions extend to non-motor realms, such as fear-based emotional processing and that these functions are supported by interactions with a wide range of brain structures. Research related to the cerebellar contributions to emotional processing has focussed primarily on the use of well-constrained conditioning paradigms in both human and non-human subjects. From these studies, cerebellar circuits appear to be critically involved in both conditioned and unconditioned responses to threatening stimuli in addition to encoding and storage of fear memory. It has been hypothesised that the computational mechanism underlying this contribution may involve internal models, where errors between actual and expected outcomes are computed within the circuitry of the cerebellum. From a clinical perspective, cerebellar abnormalities have been consistently linked to neurodevelopmental disorders, including autism. Importantly, atypical adaptive behaviour and heightened anxiety are also common amongst autistic individuals. In this review, we provide an overview of the current anatomical, physiological and theoretical understanding of cerebellar contributions to fear-based emotional processing to foster further insights into the neural circuitry underlying emotional dysregulation observed in people with autism.

The light-harvesting chlorophyll a/b-binding proteins of photosystem II family members are responsible for temperature sensitivity and leaf color phenotype in albino tea plant.

INTRODUCTION: Light-harvesting chlorophyll a/b-binding (LHCB) protein complexes of photosystem II are integral to the formation of thylakoid structure and the photosynthetic process. They play an important role in photoprotection, a crucial process in leaf development under low-temperature stress. Nonetheless, potential key genes directly related to low-temperature response and albino phenotype have not been precisely identified in tea plant. Moreover, there are no studies simultaneously investigating multiple albino tea cultivars with different temperature sensitivity. OBJECTIVES: The study aimed to clarify the basic characteristics of CsLHCB gene family members, and identify critical CsLHCB genes potentially influential in leaf color phenotypic variation and low-temperature stress response by contrasting green and albino tea cultivars. Concurrently, exploring the differential expression of the CsLHCB gene family across diverse temperature-sensitive albino tea cultivars. METHODS: We identified 20 putative CsLHCB genes according to phylogenetic analysis. Evolutionary relationships, gene duplication, chromosomal localization, and structures were analyzed by TBtools; the physiological and biochemical characteristics were analyzed by protein analysis websites; the differences in coding sequences and protein accumulation in green and albino tea cultivars, gene expression with maturity were tested by molecular biology technology; and protein interaction was analyzed in the STRING database. RESULTS: All genes were categorized into seven groups, mapping onto 7 chromosomes, including three tandem and one segmental duplications. They all own a conserved chlorophyll A/B binding protein domain. The expression of CsLHCB genes was tissue-specific, predominantly in leaves. CsLHCB5 may play a key role in the process of leaf maturation and senescence. In contrast to CsLHCB5, CsLHCB1.1, CsLHCB2, and CsLHCB3.2 were highly conserved in amino acid sequence between green and albino tea cultivars. In albino tea cultivars, unlike in green cultivars, the expression of CsLHCB1.1, CsLHCB1.2, and CsLHCB2 was down-regulated under low-temperature stress. The accumulation of CsLHCB1 and CsLHCB5 proteins was lower in albino tea cultivars. Greater accumulation of CsLHCB2 protein was detected in RX1 and RX2 compared to other albino cultivars. CONCLUSIONS: CsLHCB1.1, CsLHCB1.2, and CsLHCB2 played a role in the response to low-temperature stress. The amino acid sequence site mutation of CsLHCB5 would distinguish the green and albino tea cultivars. The less accumulation of CsLHCB1 and CsLHCB5 had a potential influence on albino leaves. Albino cultivars more sensitive to temperature exhibited lower CsLHCB gene expression. CsLHCB2 may serve as an indicator of temperature sensitivity differences in albino tea cultivars. This study could provide a reference for further studies of the functions of the CsLHCB family and contribute to research on the mechanism of the albino in tea plant.

GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells.

BACKGROUND: The pulmonary surfactant that lines the air-liquid surface within alveoli is a protein-lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. METHOD: GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. RESULTS: Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS-Erk-Foxo1-Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. CONCLUSIONS: In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Comparative analysis of clinicopathologic characteristics and prognosis between nasal and nonnasal extranodal NK/T-cell lymphoma.

BACKGROUND: The clinicopathologic characteristics and prognosis of nasal and nonnasal extranodal natural killer T-cell lymphoma (ENKTL) are considered to be different. However, the underlying features responsible for these differences are not well clarified especially in the era of asparaginase therapy. METHODS: In total, 1007 newly diagnosed ENKTL patients from 11 medical centers were included in this study. Clinicopathologic characteristics and survival data were collected. The chi-squared test and Kruskal-Wallis test were utilized for the comparison of different groups. Univariable and multivariable Cox proportional hazards models were used to screen prognostic factors. RESULTS: Overall, 869 (86.3%) patients were nasal forms. Compared to patients with nasal ENKTL, nonnasal patients were at more advanced stages and had poor performance status, bone marrow involvement, elevated serum lactate dehydrogenase (LDH), and CD56-negative status (p < 0.05). The 5-year overall survival (OS) for nasal and nonnasal patients were 65.6% and 45.0%, respectively. The OS of nasal forms patients were superior to nonnasal patients, especially in Eastern Cooperative Oncology Group performance status (ECOG PS) (≥2), advanced stage, KPI (HIR/HR), IPI (HIR/HR), PINK (HR), and high EBV DNA load groups. In patients treated with pegaspargase/L-asparaginase-based regimens, the OS of nasal patients was better than that of nonnasal patients. After adjusting the covariates of age, stage, ECOG PS score, LDH, B symptoms, and BM involvement, results showed that the nonnasal site was associated with poor survival of ENKTL. CONCLUSIONS: The clinicopathologic characteristics and prognosis of nasal and nonnasal ENKTL patients are different. Nasal forms patients had superior OS than nonnasal patients, especially in the era of asparaginase.

Homodimer-mediated phosphorylation of C/EBPα-p42 S16 modulates acute myeloid leukaemia differentiation through liquid-liquid phase separation.

CCAAT/enhancer binding protein α (C/EBPα) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. Here, we show that C/EBPα regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPα-p30. Considering that C/EBPα-p30 inhibits the functions of C/EBPα through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPα-p42 was constructed, and dynamic inhibition of C/EBPα phase separation was observed, demonstrating the importance of C/EBPα-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPα-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPα-p30/C/EBPα-p42 ratio rescued the phase separation of C/EBPα in AML cells, which provided a new insight for the treatment of the AML.