Functional Assays Research Articles

Damping excessive viral-induced IFN-γ rescues the impaired anti-Aspergillus host immune response in influenza-associated pulmonary aspergillosis

Influenza-associated pulmonary aspergillosis (IAPA) is a severe fungal superinfection in critically ill influenza patients that is of incompletely understood pathogenesis. Despite the use of contemporary therapies with antifungal and antivirals, mortality rates remain unacceptably high. We aimed to unravel the IAPA immunopathogenesis as a means to develop adjunctive immunomodulatory therapies. We used a murine model of IAPA to investigate how influenza predisposes to the development of invasive pulmonary aspergillosis. Immunocompetent mice were challenged with an intranasal instillation of influenza on day 0 followed by an orotracheal inoculation with Aspergillus 4 days later. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (μCT) and fungal burden with bioluminescence imaging (BLI). At endpoint, high parameter immunophenotyping, spatial transcriptomics, histopathology, dynamic phagosome biogenesis assays with live imaging, immunofluorescence staining, specialized functional phagocytosis and killing assays were performed. We uncovered an early exuberant influenza-induced interferon-gamma (IFN-γ) production as the major driver of immunopathology in IAPA and delineated the molecular mechanisms. Specifically, excessive IFN-γ production resulted in a defective Th17-immune response, depletion of macrophages, and impaired killing of Aspergillus conidia by macrophages due to the inhibition of NADPH oxidase-dependent activation of LC3-associated phagocytosis (LAP). Markedly, mice with partial or complete genetic ablation of IFN-γ had a restored Th17-immune response, LAP-dependent mechanism of killing and were fully protected from invasive fungal infection. Together, these results identify exuberant viral induced IFN-γ production as a major driver of immune dysfunction in IAPA, paving the way to explore the use of excessive viral-induced IFN-γ as a biomarker and new immunotherapeutic target in IAPA. This research was funded by the Research Foundation Flanders (FWO), project funding under Grant G053121N to JW, SHB and GVV; G057721N, G0G4820N to GVV; 1506114N to KL and GVV; KU Leuven internal funds (C24/17/061) to GVV, clinical research funding to JW, Research Foundation Flanders (FWO) aspirant mandate under Grant 1186121N/1186123N to LS, 11B5520N to FS, 1SF2222N to EV and 11M6922N/11M6924N to SF, travel grants V428023N, K103723N, K217722N to LS. FLvdV was supported by a Vidi grant of the Netherlands Association for Scientific Research. FLvdV, JW, AC and GC were supported by the Europeans Union's Horizon 2020 research and innovation program under grant agreement no 847507 HDM-FUN. AC was also supported by the Fundação para a Ciência e a Tecnologia (FCT), with the references UIDB/50026/2020, UIDP/50026/2020, PTDC/MED-OUT/1112/2021 (https://doi.org/10.54499/PTDC/MED-OUT/1112/2021), and 2022.06674.PTDC (http://doi.org/10.54499/2022.06674.PTDC); and the "la Caixa" Foundation under the agreement LCF/PR/HR22/52420003 (MICROFUN).

A glutathione S-transferase PcGSTMu2 involved in the detoxification of bifenazate in Panonychus citri.

The citri red mite, Panonychus citri (McGregor), is an important citrus pest worldwide, causing enormous economic losses to citrus production. Bifenazate is a widely used acaricide for controlling P. citri. The detoxification mechanism of bifenazate is not clear in P. citri. PcGSTMu2, a significantly upregulated GST gene, was identified by the transcriptome analysis of P. citri after bifenazate exposure. The expression level of PcGSTMu2 was significantly increased after bifenazate exposure. By using RNAi of PcGSTMu2, the susceptibility of P. citri to bifenazate was significantly increased. Protein modeling and docking of PcGSTMu2 with GSH and bifenazate indicated the potential amino acid residues for binding in the active site. Heterologous expression and in vitro functional assays further revealed that PcGSTMu2 could deplete bifenazate. These results indicated that PcGSTMu2 plays an important role in the detoxification of bifenazate in P. citri and provides the molecular foundation for understanding bifenazate metabolism in P. citri. © 2024 Society of Chemical Industry.

Genome-wide identification and comprehensive analysis of the AP2/ERF gene family in Prunus sibirica under low-temperature stress

BackgroundAP2/ERF transcription factors are involved in the regulation of growth, development, and stress response in plants. Although the gene family has been characterized in various species, such as Oryza sativa, Arabidopsis thaliana, and Populus trichocarpa, studies on the Prunus sibirica AP2/ERF (PsAP2/ERF) gene family are lacking. In this study, PsAP2/ERFs in P. sibirica were characterized by genomic and transcriptomic analyses.ResultsIn the study, 112 PsAP2/ERFs were identified and categorized into 16 subfamilies. Within each subfamily, PsAP2/ERFs exhibited similar exon-intron structures and motif compositions. Additionally, 50 pairs of segmentally duplicated genes were identified within the PsAP2/ERF gene family. Our experimental results showed that 20 PsAP2/ERFs are highly expressed in leaves, roots, and pistils under low-temperature stress conditions. Among them, the expression of PsAP2/ERF21, PsAP2/ERF56 and PsAP2/ERF88 was significantly up-regulated during the treatment period, and it was hypothesised that members of the PsAP2/ERF family play an important role inlow temperature stress tolerance.ConclusionsThis study improves our understanding of the molecular basis of development and low-temperature stress response in P. sibirica and provides a solid scientific foundation for further functional assays and evolutionary analyses of PsAP2/ERFs.

The oncogenic ADAMTS1–VCAN–EGFR cyclic axis drives anoikis resistance and invasion in renal cell carcinoma

BackgroundMetastasis, the leading cause of renal cell carcinoma (RCC) mortality, involves cancer cells resisting anoikis and invading. Until now, the role of the matrix metalloproteinase (MMP)-related enzyme, A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1), in RCC anoikis regulation remains unclear.MethodsThe clinical significance of ADAMTS1 and its associated molecules in patients with RCC was investigated using data from the Gene Expression Omnibus (GEO) and TCGA datasets. Human phosphoreceptor tyrosine kinase (RTK) array, luciferase reporter assays, immunoprecipitation (IP) assays, western blotting, and real-time reverse-transcription quantitative polymerase chain reaction (RT–qPCR) were used to elucidate the underlying mechanisms of ADAMTS1. Functional assays, including anoikis resistance assays, invasion assays, and a Zebrafish xenotransplantation model, were conducted to assess the roles of ADAMTS1 in conferring resistance to anoikis in RCC.ResultsThis study found elevated ADAMTS1 transcripts in RCC tissues that were correlated with a poor prognosis. ADAMTS1 manipulation significantly affected cell anoikis through the mitochondrial pathway in RCC cells. Human receptor tyrosine kinase (RTK) array screening identified that epidermal growth factor receptor (EGFR) activation was responsible for ADAMTS1-induced anoikis resistance and invasion. Further investigations revealed that enzymatically active ADAMTS1-induced versican V1 (VCAN V1) proteolysis led to EGFR transactivation, which in turn, through positive feedback, regulated ADAMTS1. Additionally, ADAMTS1 can form a complex with p53 to influence EGFR signaling. In vivo, VCAN or EGFR knockdown reversed ADAMTS1-induced prometastatic characteristics of RCC. A clinical analysis revealed a positive correlation between ADAMTS1 and VCAN or the EGFR and patients with RCC with high ADAMTS1 and VCAN expression had the worst prognoses.ConclusionsOur results collectively uncover a novel cyclic axis involving ADAMTS1–VCAN–EGFR, which significantly contributes to RCC invasion and resistance to anoikis, thus presenting a promising therapeutic target for RCC metastasis.

Characterization and angiogenic potential of CD146+ endometrial stem cells

BackgroundThe human endometrium, lining the inner uterus, regenerates over 400 times uniquely during a woman’s reproductive life. Endometrial stem cells (eSCs) enrich the tissue, resulting in a dense vascular network, significant angiogenic potential, and effective regeneration power. Being of natural angiogenic properties and proven effective in the treatment of vascular disorders, eSCs can be considered safe, reliable, and superior to other post-natal stem cells. Cluster of Differentiation 146 (CD146) has emerged as a pivotal marker associated with pericytes and endothelial cells for promoting angiogenesis. Endometrial cells with high CD146 expression could proliferate and differentiate into multiple lineages. This study will explore the role of CD146 in eSCs, focusing on the potential to boost the angiogenic and regenerative functions of the cells. The novelty of this study lies in the investigation of CD146 on eSC function, which may open new possibilities for eSC-based therapy in regenerative medicine and vascular disorders.MethodsThe study involved obtaining endometrial biopsies from active reproducing women to isolate and cultivate eSCs. eSCs were assessed for growth factor secretion pattern, characterized for their mesenchymal properties. Finally, eSCs were tested for their angiogenic potential by angiogenic gene expression profile and in-ovo chick embryo model. As aimed, to check the role of CD146 in eSC angiogenesis, CD146+ cells were magnetically sorted and cultured. The sorted cells underwent various analyses, including flowcytometry to identify mesenchymal markers and human growth factor panel to analyze growth factor secretion profiles The study evaluated the angiogenic potential of CD146 + cells using functional assays, including ring formation, endothelial differentiation, and wound scratch assays, to evaluate cell migration and healing capabilities. Molecular insights were obtained through chemokine and cytokine investigations In-ovo Chick model assay was conducted to check the angiogenic potential and evaluated through macroscopic as well as through immunohistochemistry.ResultEndometrial stem cells (eSCs) were successfully isolated using a combination of mechanical and enzymatic digestion, followed by culturing in complete DMEM media. The secretion profile of eSCs revealed significant production of various angiogenic growth factors, including Granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Vascular endothelial growth factor (VEGF), Fibroblast growth factors (FGF), and Platelet derived growth factor AA (PDGF-AA). The angiogenic gene profile indicated upregulation of several angiogenic genes in eSCs. The mesenchymal nature of eSCs was demonstrated through surface marker analysis (Cluster of differentiation 73, Cluster of differentiation 90, Cluster of differentiation 105) and trilineage differentiation. The in-ovo chick model confirmed the angiogenic potential of eSCs. CD146+ cells, isolated via magnetic sorting, exhibited enhanced angiogenic potential. These cells secreted significant levels of angiogenic growth factors such as VEGF. In Matrigel assays, CD146+ cells formed endothelial ring structures more rapidly and persistently than unsorted eSCs. Semi-quantitative PCR confirmed their endothelial differentiation. CD146+ cells express various angiogenic chemokines such as CXCL5, CXCL8, CCL3, and CCL20 and cytokines such as GM-CSF, Interleukin-1β (IL-1β), Interleukin-6 (IL-6), PDGF AA/BB, Epidermal growth factor (EGF), Endothelin 1, Angiopoietin. In-ovo chick model assay showed that CD146+ cells had superior angiogenesis, with more nodes, junctions, and segments compared to eSCs and controls. Immunohistochemistry confirmed increased expression of endothelial markers (Cluster of differentiation 31, VEGF, Vascular associated protein (VAP), Von Willebrand factor (vWF) in CD146+ cells.ConclusionThe study highlights the angiogenic potential of endometrial stem cells, particularly the CD146+ cell population. These cells promote angiogenesis, secreting growth factors and forming stable blood vessel structures. CD146+ cells have higher expression levels of VEGF and TGF-α, key factors in angiogenesis. This suggests CD146+ eSCs may be promising for therapeutic applications in vascular diseases requiring angiogenesis. Further research is needed.

Uncovering recessive alleles in rare Mendelian disorders by genome sequencing of 174 individuals with monoallelic pathogenic variants.

Clinical exome sequencing (ES) has facilitated genetic diagnosis in individuals with a rare genetic disorder by analysis of all protein-coding sequences in a single experiment. However, in 40-60% of patients, a conclusive diagnosis remains elusive. In 2-5% of these individuals, ES does identify a disease-associated monoallelic variant in a recessive disorder. We hypothesized that short-read genome sequencing (GS) might uncover a pathogenic variant on the second allele, thereby increasing diagnostic yield. We performed GS for 174 individuals in whom ES identified a monoallelic pathogenic variant in a gene associated with recessive disease related to their phenotype. GS interpretation was limited to the (non-)coding parts of the gene in which this first pathogenic variant was identified, focusing on splice-disrupting variants. Firstly, we uncovered a second pathogenic variant affecting coding sequence in five individuals, including two SNV/indel variants, two copy number variants, and one insertion. Secondly, for 24 individuals, we identified a total of 31 rare non-coding intronic SNV/indel variants, all predicted to disrupt splicing. Using functional follow-up assays, we confirmed an effect on splicing for three of these variants (in ABCA4, POLR3A and COL4A4) in three individuals. In summary, we identified a (likely) pathogenic second variant in 4.6% (8/174), and a possible diagnosis for 12.1% (21/174) of our cohort. Hence, when performing GS as first-tier diagnostic test, including the interpretation of SVs and rare intronic variants in known recessive disease genes, the overall diagnostic yield of rare disease will increase. The added diagnostic value of GS for recessive disease In our cohort of 174 individuals (84 males and 90 females) with a monoallelic pathogenic variant in genes associated with a wide and diverse range of recessive diseases (pie chart), using genome sequencing (GS) and a systematic approach (methods), we identified eight new diagnoses (4.6%). We identified a second likely pathogenic variant in eight individuals (results); In two a second coding variant was found, in three others, a rare non-coding SNV anticipated to disrupt splicing was uncovered, and in three individuals a structural rearrangement was identified (two copy number variants (CNV), and one structural variant (SV)).

Rare genetic variants of NLRP12 in Admixed Latino-American Children with SARS-CoV-2-related Multisystem Inflammatory Syndrome.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare, potentially fatal complication of SARS-CoV-2 infection. Genetic defects in inflammation-related pathways have been linked to MIS-C, but additional research is needed, especially in diverse ethnic groups. The present study aimed to identify genetic variants underlying MIS-C in Brazilian patients. Whole-exome sequencing was performed, focusing on genes involved in the host immune response to SARS-CoV-2. Functional assays assessed the impact of selected variants on NF-κB signaling. Nine rare, potentially deleterious variants were found in eight of 21 patients, located in IL17RC, IFNA10, or NLRP12 genes. Unlike the wild-type NLRP12 protein, which inhibits NF-κB activation in HEK 293T cells, the mutant NLRP12 proteins have significantly reduced inhibitory properties. In conclusion, our results indicate that rare autosomal variants in immune-related genes may underlie MIS-C, highlighting the potential role of NLRP12 in its predisposition. These findings provide new insights for the appropriate management of MIS-C.

A human-serum-free medium can induce more infectious P. falciparum gametocytes than a conventional human-serum-containing medium

Malaria remains a global health problem, and the standard membrane feeding assay (SMFA) is a key functional assay for development of new interventions to stop malaria transmission from human to mosquito. For SMFA, media with ~ 10% of human serum has been used for infectious gametocyte cultures, however, there are multiple challenges to obtain a suitable human serum. Here we show a human-serum-free culture medium (HSF), which was a mixture of two stem cell culture media and AlbuMAX, supported infectious gametocyte growth. Moreover, the HSF-induced gametocytes elicited significantly higher numbers of oocysts compared to gametocytes cultured with conventional human serum medium (Conv). While some caution is required when comparing percent transmission reducing activity data generated from HSF-SMFA and Conv-SMFA, the HSF method can facilitate the establishment of gametocyte cultures or SMFA by bypassing the need for human serum. Thus, this study will support future development of P. falciparum transmission-blocking interventions.

Monoallelic loss of RB1 Enhances Osteogenic Differentiation and Delays DNA Repair Without Inducing Tumorigenicity

The Retinoblastoma (RB1) gene plays a pivotal role in osteogenic differentiation. Our previous study, employing temporal gene expression analysis using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), revealed the deregulation of osteogenic differentiation in patient-derived heterozygous RB1 mutant orbital adipose-derived mesenchymal stem cells (OAMSCs). The study revealed increased Alizarin Red staining, suggesting heightened mineralization without a corresponding increase in osteogenic lineage-specific gene expression. In this study, we performed high-throughput RNA sequencing on RB1+/+ and RB1+/- patient-derived OAMSCs differentiated towards the osteogenic lineage to investigate the pathways and molecular mechanisms. The pathway analysis revealed significant differences in cell proliferation, DNA repair, osteoblast differentiation, and cancer-related pathways in RB1+/- OAMSC-derived osteocytes. These findings were subsequently validated through functional assays. The study revealed that osteogenic differentiation is increased in RB1+/- cells, along with enhanced proliferation of the osteocytes. There were delayed but persistent DNA repair mechanisms in RB1+/- osteocytes, which were sufficient to maintain genomic integrity, thereby preventing or delaying the onset of tumors. This contrasts with our earlier observation of increased mineralization without corresponding gene expression changes, emphasizing the importance of high-throughput analysis over preselected gene set analysis in comprehending functional assay results.

Permeation mechanisms of hydrogen peroxide and water through Plasma Membrane Intrinsic Protein aquaporins.

Hydrogen peroxide (H2O2) transport by aquaporins (AQP) is a critical feature for cellular redox signaling. However, the H2O2 permeation mechanism through these channels remains poorly understood. Through functional assays, two Plasma membrane Intrinsic Protein (PIP) AQP from Medicago truncatula, MtPIP2;2 and MtPIP2;3 have been identified as pH-gated channels capable of facilitating the permeation of both water (H2O) and H2O2. Employing a combination of unbiased and enhanced sampling molecular dynamics simulations, we investigated the key barriers and translocation mechanisms governing H2O2 permeation through these AQP in both open and closed conformational states. Our findings reveal that both H2O and H2O2 encounter their primary permeation barrier within the selectivity filter (SF) region of MtPIP2;3. In addition to the SF barrier, a second energetic barrier at the NPA (asparagine-proline-alanine) region that is more restrictive for the passage of H2O2 than for H2O, was found. This behavior can be attributed to a dissimilar geometric arrangement and hydrogen bonding profile between both molecules in this area. Collectively, these findings suggest mechanistic heterogeneity in H2O and H2O2 permeation through PIPs.

ACTL8 Promotes the Progression of Gastric Cancer Through PI3K/AKT/mTOR Signaling Pathway.

Actin-like protein 8 (ACTL8) significantly correlates with tumor growth and prognosis across various cancer types. Nevertheless, the potential relationship between ACTL8 and gastric cancer (GC) remains uncertain. This study aimed to elucidate the role of ACTL8 in human GC cells and to explore its mechanism. Bioinformatics analysis tools, such as GEPIA2, Kaplan-Meier, and STRING, were utilized for a comprehensive investigation of the characteristics and functional roles of ACTL8 in GC, including differential expression, prognostic value, and related signaling pathways. Subsequently, gene expression analyses, cell function assays, and signaling pathway experiments were conducted to verify key findings. Bioinformatics analysis showed that ACTL8 was significantly elevated in GC and closely associated with poor prognosis. Gene expression experiments confirmed the bioinformatics results. Furthermore, ACTL8 knockdown markedly reduced GC cell proliferation and inhibited migration and invasion. Mechanistically, a significant increase in the phosphorylation levels of signaling proteins was observed in GC cells following ACTL8 overexpression,and PI3K/Akt/mTOR pathway inhibitors could reverse this effect. ACTL8 expression is significantly upregulated in GC cells and is closely correlated with poor patient prognosis. Further mechanistic studies revealed that ACTL8 may promote GC cell migration and proliferation through activation of the PI3K/Akt/mTOR signaling pathway. Consequently, ACTL8 emerges as a promising therapeutic target for GC.

Assays of Variant Effect and Automated Patch Clamping Improve KCNH2-LQTS Variant Classification and Cardiac Event Risk Stratification.

Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2. Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and adjusted QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76-0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83-0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81-0.88]). High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

Agrobacterium-mediated transient expression in Torreya grandis cones: A simple and rapid tool for gene expression and functional gene assay

Agrobacterium-mediated transient gene expression is a powerful tool for characterizing gene function in plants, particularly in fruit trees with long juvenile periods. However, few studies have systematically explored transient expression in nut trees, such as T. grandis. In this study, we developed a rapid and easily manipulated transient transformation system using Agrobacterium-mediated injection infiltration in T. grandis cones. Cones at the enlargement stage (30 days after aril protrusion) were used, and the bacterial solution was gently injected into the kernel at a depth of 1 cm, filling nearly the entire cone, as indicated by a red dye. Using the green fluorescent protein (GFP) gene as a reporter for Agrobacterium-mediated transformation, we confirmed GFP expression in T. grandis cone successfully through confocal microscopy, western blot, and PCR. Transformation conditions, including bacterial density and days after agroinfiltration, were optimized for higher transformation efficiency, with the optimal conditions being an OD600 of 0.6 and an agroinfiltration period of 6 - 9 days. Transient overexpression of TgWRKY47, a cell size regulator, altered cell size in T. grandis cones, indicating that this Agrobacterium infiltration method can be used for gene functional analysis. Furthermore, confocal microscopy revealed that TgWRKY47-GFP was localized in the nucleus along with a nuclear marker, demonstrating its applicability for protein subcellular localization. In summary, this method presents a versatile tool for efficient and targeted transient gene functional analysis and subcellular localization in T. grandis cones, offering new insights into nut tree biology research.

Exosome-like Nanoparticles, High in Trans-δ-Viniferin Derivatives, Produced from Grape Cell Cultures: Preparation, Characterization, and Anticancer Properties.

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer.

The Effects of the Pneumonia Lung Microenvironment on MSC Function.

Despite promise in preclinical models of acute respiratory distress syndrome (ARDS), mesenchymal stem cells (MSC) have failed to translate to therapeutic benefit in clinical trials. The MSC is a live cell medicine and interacts with the patient's disease state. Here, we explored this interaction, seeking to devise strategies to enhance MSC therapeutic function. Human bone-marrow-derived MSCs were exposed to lung homogenate from healthy and E. coli-induced ARDS rat models. Apoptosis and functional assays of the MSCs were performed. The ARDS model showed reduced arterial oxygenation, decreased lung compliance and an inflammatory microenvironment compared to controls. MSCs underwent more apoptosis after stimulation by lung homogenate from controls compared to E. coli, which may explain why MSCs persist longer in ARDS subjects after administration. Changes in expression of cell surface markers and cytokines were associated with lung homogenate from different groups. The anti-microbial effects of MSCs did not change with the stimulation. Moreover, the conditioned media from lung-homogenate-stimulated MSCs inhibited T-cell proliferation. These findings suggest that the ARDS microenvironment plays an important role in the MSC's therapeutic mechanism of action, and changes can inform strategies to modulate MSC-based cell therapy for ARDS.

Investigating MATN3 and ASPN as novel drivers of gastric cancer progression via EMT pathways

Abstract Gastric cancer (GC) is a leading cause of cancer-related deaths globally, necessitating the identification of novel therapeutic targets. This study investigates the roles of MATN3 and ASPN in GC progression via the epithelial-mesenchymal transition (EMT) pathway. Analysis of the Cancer Genome Atlas—Stomach Adenocarcinoma (TCGA-STAD) dataset revealed that both MATN3 and ASPN are significantly upregulated in GC tissues and correlate with poor patient survival. Protein–protein interaction and co-expression analyses confirmed a direct interaction between MATN3 and ASPN, suggesting their synergistic role in EMT activation. Functional assays demonstrated that MATN3 promotes GC cell proliferation, migration, and invasion, while its knockdown inhibits these malignant behaviors and induces apoptosis. ASPN overexpression further amplified these oncogenic effects. In vivo, studies in a mouse model corroborated that co-overexpression of MATN3 and ASPN enhances tumor growth and metastasis. These findings highlight the MATN3-ASPN axis as a potential therapeutic target in GC, offering new insights into the molecular mechanisms driving GC progression.

Targeting cellular plasticity: esculetin-driven reversion of stem cell-like characteristics and EMT phenotype in transforming cells with sequential p53/p73 knockdowns

The intricate interplay of cancer stem cell plasticity, along with the bidirectional transformation between epithelial-mesenchymal states, introduces further intricacy to offer insights into newer therapeutic approaches. Differentiation therapy, while successful in targeting leukemic stem cells, has shown limited overall success, with only a few promising instances. Using colon carcinoma cell strains with sequential p53/p73 knockdowns, our study underscores the association between p53/p73 and the maintenance of cellular plasticity. Morphological alterations corresponding with cell surface marker expressions, transcriptome analysis and functional assays were performed to access stemness and EMT (Epithelial-Mesenchymal Transition) characteristics in the spectrum of cells exhibiting sequential p53 and p73 knockdowns. Notably, our investigation explores the effectiveness of esculetin in reversing the shift from an epithelial to a mesenchymal phenotype, characterized by stem cell-like traits. Esculetin significantly induces enterocyte differentiation and promotes epithelial cell polarity by altering Wnt axes in Cancer Stem Cell-like cells characterized by high mesenchymal features. These results align with our previous findings in leukemic blast cells, establishing esculetin as an effective differentiating agent in both Acute Myeloid Leukemia (AML) and solid tumor cells.

Dysfunctional High-Density Lipoprotein Cholesterol and Coronary Artery Disease: A Narrative Review.

High-density lipoprotein (HDL) cholesterol is traditionally viewed as protective against cardiovascular disease (CVD). However, emerging evidence reveals that dysfunctional HDL, characterized by impaired reverse cholesterol transport (RCT), reduced anti-inflammatory and antioxidant activities and increased endothelial dysfunction, which can contribute to coronary artery disease (CAD). Dysfunctional HDL, resulting from oxidative modifications of Apolipoprotein A-1 (Apo A-1) and enzyme inactivation, fails to effectively remove cholesterol from peripheral tissues and may promote inflammation and atherosclerosis. Genetic mutations affecting HDL metabolism further complicate its role in cardiovascular health. Studies have shown that conventional therapies aimed at raising HDL-C levels do not necessarily reduce cardiovascular events, highlighting the need for new approaches that improve HDL functionality. Therapeutic strategies such as Apo A-1 mimetic peptides, reconstituted HDL infusions, and drugs targeting specific HDL metabolic pathways are being explored. Additionally, weight loss, statin therapy, and niacin have shown potential in enhancing HDL function. The pathophysiology of dysfunctional HDL involves complex mechanisms, including oxidative stress, inflammation, and genetic mutations, which alter its structure and function, diminishing its cardioprotective effects. New functional assays, such as the cholesterol efflux capacity (CEC) and HDL inflammatory index, provide more accurate predictions of cardiovascular risk by assessing HDL quality rather than quantity. As research progresses, the focus is shifting towards therapeutic strategies that enhance HDL function and address the root causes of its dysfunction, offering a more effective approach to reducing cardiovascular risk and preventing CAD.

Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We have developed a codon optimized CRISPRi/a for zebrafish; here we provide proof-of-principle data across established pigmentary and growth phenotypes. We established a zebrafish codon-optimized cas9 gene, harboring mutations D10A and D839A to render the protein catalytically inactive ( dCas9 ). Similarly, codon-optimized Krüppel associated box (KRAB) and methylated CP2 (MeCP2) inactivating domains or VP64 activator domain were cloned downstream from dCas9 for CRISPRi and CRISPRa, respectively. To validate CRISPRi, we targeted key genes in melanocyte differentiation ( sox10, mitfa, and mitfb) ; and melanin production (tyrosinase; tyr ). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5'-UTR resulted in larvae with hypopigmented epidermal melanocytes. Transcription factors mitfa and mitfb similarly direct differentiation and pigmentation of epidermal melanocytes ( mitfa ) and retinal pigment epithelium (RPE, mitfb ). CRISPRi-mediated targeting of their promoters or 5'-UTR also results in pronounced hypopigmentation of epidermal melanocytes ( mitfa ), and RPE ( mitfb ). So too, targeting CRISPRi to the sox10 promoter results in hypopigmentation of both epidermal melanocytes and RPE consistent with its role upstream of mitfa and mitfb , and tyr . Finally, we asked whether CRISPRi and CRISPRa could be used to modulate a single gene, to yield hypomorphic and hypermorphic effects, selecting mrap2a , as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene ( mc4r ). We demonstrate that targeting the mrap2a 5'-UTR with CRISPRa or CRISPRi significantly increases or decreases larval body length, respectively. We demonstrate the utility of CRISPRi/a for modulating control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.

Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection.

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on ∼1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.