CRISPR/Cas9-mediated generation of a mutant library of cotton CDPK gene family for identifying insect-resistant genes.

Calcium-dependent protein kinases (CDPKs) are pivotal signaling transduction enzymes in plants, especially responsive to diverse stress, including herbivory. In this study, through comprehensive analysis of CDPK gene family in upland cotton, we showed that GhCPKs are widely expressed in multiple tissues of cotton and positively respond to various biotic and abiotic stress. We developed a strategy for screening insect-resistant genes based on the CRISPR/Cas9 mutant library of GhCPKs. The library contains 82 members of the GhCPKs using 246 sgRNAs to generate 518 independent T0 plants. The coverage rate of target genes reached to 86.18%, the genome editing rate reached to 89.49%, and the editing heritability reached 82%. Through field insect bioassay, 14 GhCPK mutants resistant or susceptible to insect were identified. The most obvious insect-resistant mutant, cpk33/74 (simultaneously knocking out the homologous genes GhCPK33 and GhCPK74), was selected for further study. Oral secretions (OS) from Spodoptera litura induced a rapid influx of Ca2+ in cpk33/74 leaves, resulting in a significant increase in jasmonic acid (JA) content. S-adenosylmethionine synthase (SAMS) is an important protein involved in plant stress response, protein interaction experiments provided evidence of interactions between GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2, respectively. Additionally, silencing GhSAMS1 and GhSAM2 in cotton using VIGS resulted in decreased defense against S. litura. This study provides an effective strategy for constructing a mutant library of gene families in polyploid plant species and valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Atlas of metastatic gastric cancer links ferroptosis to disease progression and immunotherapy response.

BACKGROUND & AIMS: Metastases from gastric adenocarcinoma (GAC) lead to high morbidity and mortality. Developing innovative and effective therapies requires a comprehensive understanding of the tumor and immune biology of advanced GAC. Yet, collecting matched specimens from advanced, treatment-naïve GAC patients poses a significant challenge, limiting the scope of current research, which has predominantly focused on localized tumors. This gap hinders a deeper insight into the metastatic dynamics of GAC. METHODS: We performed in-depth single-cell transcriptome and immune profiling on 68 paired, treatment-naïve, primary-metastatic tumors to delineate alterations in cancer cells and their tumor microenvironment (TME) during metastatic progression. To validate our observations, we conducted comprehensive functional studies both in vitro and in vivo, employing cell lines, multiple PDX and novel mouse models of GAC. RESULTS: Liver and peritoneal metastases exhibited distinct properties in cancer cells and dynamics of TME phenotypes, supporting the notion that cancer cells and their local TMEs co-evolve at metastatic sites. Our study also revealed differential activation of cancer meta-programs across metastases. We observed evasion of cancer cell ferroptosis via GPX4 upregulation during GAC progression. Conditional depletion of Gpx4 or pharmacological inhibition of ferroptosis resistance significantly attenuated tumor growth and metastatic progression. Additionally, ferroptosis-resensitizing treatments augmented the efficacy of CAR T-cell therapy. CONCLUSIONS: This study represents the largest single-cell dataset of metastatic GACs to date. High-resolution mapping of the molecular and cellular dynamics of GAC metastasis has revealed a rationale for targeting ferroptosis defense in combination with CAR T-cell therapy as a novel therapeutic strategy with potential immense clinical implications.

Three biomarkers (HER2, PD-L1, and microsatellite status) in a large cohort of metastatic gastroesophageal adenocarcinomas: The MD Anderson Cancer Center experience.

Human epidermal growth factor receptor-2 (HER2), programmed death-ligand 1 (PD-L1), and microsatellite (MS) status are well-established biomarkers in gastroesophageal adenocarcinomas (GEAs). However, it is unclear how the combination of these biomarkers is associated with clinicopathological factors and prognosis. This retrospective study included baseline metastatic GEA patients who were tested for all three biomarkers (HER2, PD-L1, and MS status) at the MD Anderson Cancer Center between 2012 and 2022. Stratification was performed according to the combination of biomarker profiles: triple negative (TN), single positive (SP), and multiple positive (MP). Comparative analyses of clinicopathological factors and survival using combinations of biomarkers were performed. Among the 698 GEA patients analyzed, 251 (36.0%) were classified as TN, 334 (47.9%) as SP, and 113 (16.1%) as MP. The MP group showed a significant association with tumors located in the esophagus (p < .001), well to moderate differentiation (p < .001), and the absence of signet ring cells (p < .001). In the survival analysis, MP group had a significantly longer overall survival (OS) compared to the other groups (MP vs. TN, p < .001 and MP vs. SP, p < .001). Multivariate Cox regression analysis revealed that MP serves as an independent positive prognostic indicator for OS (hazard ratio = 0.63, p < .01). Our findings indicate that MP biomarkers are associated with a favorable prognosis in metastatic GEA. These results are reflective of clinical practice and offer valuable insights into how therapeutics and future biomarkers could influence therapy/prognosis.

Objective measure of binaural processing: Acoustic change complex in response to interaural phase differences.

Combining cochlear implants with binaural acoustic hearing via preserved hearing in the implanted ear(s) is commonly referred to as combined electric and acoustic stimulation (EAS). EAS fittings can provide patients with significant benefit for speech recognition in complex noise, perceived listening difficulty, and horizontal-plane localization as compared to traditional bimodal hearing conditions with contralateral and monaural acoustic hearing. However, EAS benefit varies across patients and the degree of benefit is not reliably related to the underlying audiogram. Previous research has indicated that EAS benefit for speech recognition in complex listening scenarios and localization is significantly correlated with the patients' binaural cue sensitivity, namely interaural time differences (ITD). In the context of pure tones, interaural phase differences (IPD) and ITD can be understood as two perspectives on the same phenomenon. Through simple mathematical conversion, one can be transformed into the other, illustrating their inherent interrelation for spatial hearing abilities. However, assessing binaural cue sensitivity is not part of a clinical assessment battery as psychophysical tasks are time consuming, require training to achieve performance asymptote, and specialized programming and software all of which render this clinically unfeasible. In this study, we investigated the possibility of using an objective measure of binaural cue sensitivity by the acoustic change complex (ACC) via imposition of an IPD of varying degrees at stimulus midpoint. Ten adult listeners with normal hearing were assessed on tasks of behavioral and objective binaural cue sensitivity for carrier frequencies of 250 and 1000 Hz. Results suggest that 1) ACC amplitude increases with IPD; 2) ACC-based IPD sensitivity for 250 Hz is significantly correlated with behavioral ITD sensitivity; 3) Participants were more sensitive to IPDs at 250 Hz as compared to 1000 Hz. Thus, this objective measure of IPD sensitivity may hold clinical application for pre- and post-operative assessment for individuals meeting candidacy indications for cochlear implantation with low-frequency acoustic hearing preservation as this relatively quick and objective measure may provide clinicians with information identifying patients most likely to derive benefit from EAS technology.

SMARCA4 Mutations in Gastroesophageal Adenocarcinoma: An Observational Study via a Next-Generation Sequencing Panel.

BACKGROUND: The clinical impact of SMARCA4 mutations (SMARCA4ms) in gastroesophageal adenocarcinoma (GEA) remains underexplored. This study aimed to examine the association of SMARCA4ms with clinical outcomes and co-occurrence with other gene mutations identified through a next-generation sequencing (NGS) panel in GEA patients. METHODS: A total of 256 patients with metastatic or recurrent GEA who underwent NGS panel profiling at the MD Anderson Cancer Center between 2016 and 2022 were included. Comparative analyses were performed to assess clinical outcomes related to SMARCA4ms. The frequency and types of SMARCA4ms and their co-occurrence with other gene mutations were also examined. RESULTS: SMARCA4ms were identified in 19 patients (7.4%). These SMARCA4ms were significantly associated with non-signet ring cell subtype (p = 0.044) and PD-L1 positive expression (p = 0.046). No difference in survival between the SMARCA4m and SMARCA4-normal group was observed (p = 0.84). There were significant associations between SMARCA4ms and FANCA, IGF1R, KRAS, FANCL, and PTEN alterations. Notably, 15 of the 19 SMARCA4m cases involved SNV missense mutations, with frequent co-occurrences noted with TP53, KRAS, ARID1A, and ERBB2 mutations. CONCLUSIONS: These results serve as the first comprehensive examination of the relationship between SMARCA4ms and clinical outcomes in GEA.

PixRevive: Latent Feature Diffusion Model for Compressed Video Quality Enhancement.

In recent years, the rapid prevalence of high-definition video in Internet of Things (IoT) systems has been directly facilitated by advances in imaging sensor technology. To adapt to limited uplink bandwidth, most media platforms opt to compress videos to bitrate streams for transmission. However, this compression often leads to significant texture loss and artifacts, which severely degrade the Quality of Experience (QoE). We propose a latent feature diffusion model (LFDM) for compressed video quality enhancement, which comprises a compact edge latent feature prior network (ELPN) and a conditional noise prediction network (CNPN). Specifically, we first pre-train ELPNet to construct a latent feature space that captures rich detail information for representing sharpness latent variables. Second, we incorporate these latent variables into the prediction network to iteratively guide the generation direction, thus resolving the problem that the direct application of diffusion models to temporal prediction disrupts inter-frame dependencies, thereby completing the modeling of temporal correlations. Lastly, we innovatively develop a Grouped Domain Fusion module that effectively addresses the challenges of diffusion distortion caused by naive cross-domain information fusion. Comparative experiments on the MFQEv2 benchmark validate our algorithm's superior performance in terms of both objective and subjective metrics. By integrating with codecs and image sensors, our method can provide higher video quality.

Comprehensive analysis of MAPK gene family in upland cotton (Gossypium hirsutum) and functional characterization of GhMPK31 in regulating defense response to insect infestation.

KEY MESSAGE: The transcriptomic, phenotypic and metabolomic analysis of transgenic plants overexpressing GhMPK31 in upland cotton revealed the regulation of H2O2 burst and the synthesis of defensive metabolites by GhMPK31. Mitogen-activated protein kinases (MAPKs) are a crucial class of protein kinases, which play an essential role in various biological processes in plants. Upland cotton (G. hirsutum) is the most widely cultivated cotton species with high economic value. To gain a better understanding of the role of the MAPK gene family, we conducted a comprehensive analysis of the MAPK gene family in cotton. In this study, a total of 55 GhMPK genes were identified from the whole genome of G. hirsutum. Through an investigation of the expression patterns under diverse stress conditions, we discovered that the majority of GhMPK family members demonstrated robust responses to abiotic stress, pathogen stress and pest stress. Furthermore, the overexpression of GhMPK31 in cotton leaves led to a hypersensitive response (HR)-like cell death phenotype and impaired the defense capability of cotton against herbivorous insects. Transcriptome and metabolomics data analysis showed that overexpression of GhMPK31 enhanced the expression of H2O2-related genes and reduced the accumulation of defensive related metabolites. The direct evidence of GhMPK31 interacting with GhRBOHB (H2O2-generating protein) were found by Y2H, BiFC, and LCI. Therefore, we propose that the increase of H2O2 content caused by overexpression of GhMPK31 resulted in HR-like cell death in cotton leaves while reducing the accumulation of defensive metabolites, ultimately leading to a decrease in the defense ability of cotton against herbivorous insects. This study provides valuable insights into the function of MAPK genes in plant resistance to herbivorous insects.

E-cadherin loss drives diffuse-type gastric tumorigenesis via EZH2-mediated reprogramming.

Diffuse-type gastric adenocarcinoma (DGAC) is a deadly cancer often diagnosed late and resistant to treatment. While hereditary DGAC is linked to CDH1 mutations, the role of CDH1/E-cadherin inactivation in sporadic DGAC tumorigenesis remains elusive. We discovered CDH1 inactivation in a subset of DGAC patient tumors. Analyzing single-cell transcriptomes in malignant ascites, we identified two DGAC subtypes: DGAC1 (CDH1 loss) and DGAC2 (lacking immune response). DGAC1 displayed distinct molecular signatures, activated DGAC-related pathways, and an abundance of exhausted T cells in ascites. Genetically engineered murine gastric organoids showed that Cdh1 knock-out (KO), KrasG12D, Trp53 KO (EKP) accelerates tumorigenesis with immune evasion compared with KrasG12D, Trp53 KO (KP). We also identified EZH2 as a key mediator promoting CDH1 loss-associated DGAC tumorigenesis. These findings highlight DGAC's molecular diversity and potential for personalized treatment in CDH1-inactivated patients.

Mesothelial cells with mesenchymal features enhance peritoneal dissemination by forming a protumorigenic microenvironment.

Malignant ascites accompanied by peritoneal dissemination contain various factors and cell populations as well as cancer cells; however, how the tumor microenvironment is shaped in ascites remains unclear. Single-cell proteomic profiling and a comprehensive proteomic analysis are conducted to comprehensively characterize malignant ascites. Here, we find defects in immune effectors along with immunosuppressive cell accumulation in ascites of patients with gastric cancer (GC) and identify five distinct subpopulations of CD45(-)/EpCAM(-) cells. Mesothelial cells with mesenchymal features in CD45(-)/EpCAM(-) cells are the predominant source of chemokines involved in immunosuppressive myeloid cell (IMC) recruitment. Moreover, mesothelial-mesenchymal transition (MMT)-induced mesothelial cells strongly express extracellular matrix (ECM)-related genes, including tenascin-C (TNC), enhancing metastatic colonization. These findings highlight the definite roles of the mesenchymal cell population in the development of a protumorigenic microenvironment to promote peritoneal dissemination.