Cell Biological Studies Research Articles

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Hepatocellular carcinoma hosts cholinergic neural cells and tumoral hepatocytes harboring targetable muscarinic receptors

Background & aimsUnexplained interpatient variation and treatment failure in HCC prompt for renewed approaches. Hepatic neurons, belonging to the autonomic nervous system (ANS), mediate liver/whole body cross-talks. Pathological innervation of the ANS have been identified in cancer, nurturing tumor stroma and conferring stronger carcinogenic properties. MethodsWe characterized the innervation of liver tumors from the French Liver Biobank, then applied bioinformatics to the TCGA, several other datasets and a European validation cohort, to re-evaluate patient stratification. Cell biology and pharmacology studies were also done. ResultsDensely packed nucleated DCX+, synaptophysin+, NeuN+, VAChT+, TH-, CD31-, CD45- clusters, to date undetected, were identified in human HCCs, and independently confirmed by scRNA-seq data. Using the new concept of neuronal score, human and rat HCCs displayed tightly netrin-1-associated neural reconfiguration towards cholinergic polarity along with CLD progression, cancer onset and many features of aggressive (proliferative class) HCC, including shortened survival. This score was conditioned by tumoral hepatocytes, and predicted sorafenib efficacy in the STORM HCC phase 3 trial. Conversely, intratumoral adrenergic lymphocytes were enriched in TEMRA and cytotoxic phenotypes. Amongst all cholinergic transcripts, the medically-targeted CHRM3 receptor was enriched in HCC and associated with cells’ and tumors’ pathogenic traits, displayed adverse prognostic value by the log-rank test in HCC stages 1-2, while collapsing upon experimental re-differentiation. Its pharmacological inhibition by low concentrations of anticholinergic drugs, but not cholinomimetics, decreased anchorage-independent and anoikis growth, synergized with sorafenib and lenvatinib in HCC class 1 to 3 lines, yet not in primary human hepatocytes, and preserved mature hepatocytic functions. ConclusionThese data identify cholinergic processes as instrumental in liver carcinogenesis, and support the use of EMA/FDA-approved cholinergic drugs in HCC research. Impact and implicationsHCC care has long been hampered by the enigmatic nature of disease evolution, as well as of response or resistance to treatment. Hepatic neurons are likely the least studied liver cell type and mediate patients singularities to the organ in real-time. Cholinergic inputs identified in this study as pathogenic may be targeted with the well charted pharmacopoeia of neurotropic drugs already available, for basic or clinical research purposes, with an expected high level of safety.

Increased expression of the proapoptotic presenilin associated protein is involved in neuronal tangle formation in human brain

Presenilin-associated protein (PSAP) is a mitochondrial proapoptotic protein as established in cell biology studies. It remains unknown whether it involves in neurodegenerative diseases. Here, we explored PASP expression in adult and aged human brains and its alteration relative to Alzheimer-disease (AD)-type neuropathology. In pathology-free brains, light PASP immunoreactivity (IR) occurred among largely principal neurons in the cerebrum and subcortical structures. In the brains with AD pathology, enhanced PSAP IR occurred in neuronal and neuritic profiles with a tangle-like appearance, with PSAP and pTau protein levels elevated in neocortical lysates relative to control. Neuronal/neuritic profiles with enhanced PSAP IR partially colocalized with pTau, but invariably with Amylo-Glo labelled tangles. The neuronal somata with enhanced PASP IR also showed diminished IR for casein kinase 1 delta (Ck1δ), a marker of granulovacuolar degeneration; and diminished IR for sortilin, which is normally expressed in membrane and intracellular protein sorting/trafficking organelles. In old 3xTg-AD mice with β-amyloid and pTau pathologies developed in the brain, PSAP IR in the cerebral sections exhibited no difference relative to wildtype mice. These findings indicate that PSAP upregulation is involved in the course of tangle formation especially in the human brain during aging and in AD pathogenesis.

The genome and comparative transcriptome of the euryhaline model ciliate Paramecium duboscqui reveal adaptations to environmental salinity

BackgroundAs a potential model organism for studies of environmental and cell biology, Paramecium duboscqui is a special euryhaline species of Paramecium that can be found in fresh, brackish, or marine water in natural salinity ranges between 0‰ and 33‰. However, the genome information as well as molecular mechanisms that account for its remarkable halotolerant traits remain extremely unknown. To characterize its genome feature, we combined PacBio and Illumina sequencing to assemble the first high-quality and near-complete macronuclear genome of P. duboscqui. Meanwhile, comparative transcriptomic profiles under different salinities gave underlying insight into the molecular mechanism of its adaptations to environmental salinity.ResultsThe results showed that the MAC genome of P. duboscqui comprises 160 contigs, with 113 of them possessing telomere (~ 28.82 Mb haploid genome size). Through comparative genomic analyses with the other ciliate, we found that gene families encoding transmembrane transporter proteins have been expanded in P. duboscqui, showing enormous potential in salinity adaptation. Like other Paramecium, P. duboscqui utilizes TGA as its only termination codon and has reassigned TAA and TAG to encode glutamine. P. duboscqui showed different growth rates under different salinities, with an optimum growth rate in 5‰ salinity. A comparison of the transcriptomic profiles among P. duboscqui grown under different concentrations showed that genes involved in protein folding, oxygen respiration, and glutathione-dependent detoxification were upregulated in the high-salt group, whereas genes encoding DNA-binding proteins and transcription factors were upregulated in the low-salt group, suggesting distinct mechanisms for responding to low and high salinity. Weighted gene coexpression network analysis (WGCNA) linked the hub genes expressed at 30‰ salinity to cysteine-type peptidase activity, lipid transfer, sodium hydrogen exchange, and cell division, with the hub genes expressed at 0‰ salinity involved in transmembrane transport and protein localization.ConclusionsThis study characterizes a new euryhaline model Paramecium, provides novel insights into Paramecium evolution, and describes the molecular mechanisms that have allow P. duboscqui to adapt to different osmotic environments.

An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development

BackgroundCucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop.ResultsPollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods.ConclusionThrough parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique’s power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

Suppression of B-type natriuretic peptide gene expression in cardiomyocytes under anoxic conditions

Several cell biology studies have focused on the effects of hypoxic environments on cardiomyocytes. However, the effect of anoxic conditions on cardiomyocytes remains largely unexplored. In the present study, we investigated the direct effects of anoxia on B-type natriuretic peptide (BNP) gene expression in cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were exposed to anoxia using an airtight chamber saturated with 95% N2/5% CO2. BNP mRNA levels in NRCM were substantially reduced after more than 8hours of anoxia exposure, whereas after reoxygenation, BNP gene expression levels recovered in a time-dependent manner and significantly increased after 24hours of reoxygenation. BNP mRNA levels suppressed under anoxic conditions were significantly increased by aldosterone-induced activation of sodium-proton exchanger 1 (NHE1), which was cancelled by an NHE1 inhibitor, suggesting that anoxia reduces BNP gene expression, at least in part, in an NHE1-dependent manner. In summary, we found that BNP gene expression in cardiomyocytes decreases under anoxic conditions, in contrast to previous research findings that BNP expression increases under hypoxic conditions. These findings reveal a new insight that, within a single heart tissue in various cardiovascular diseases, such as myocardial infarction, the biological responses of cardiomyocytes are fundamentally different in regions of anoxia and hypoxia.

DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway.

Emission based response of Er3+, Tb3+, and Er3+-Tb3+ co-doped 1393 bioactive glasses along with HPTS towards CO2

The HPTS serves as a versatile tool in pH and CO2 sensing studies, as well as in cell biology studies of various processes. However, the long-term photostability of HPTS as a fluorescent dye is an important consideration in its use, especially for long-term applications. In this work, sol–gel synthesized Er3+, Tb3+ and Er3+: Tb3+ co-doped 1393 bioactive glass particles were incorporated with the HPTS in the presence of ionic liquid in an ethyl cellulose matrix. The interaction of the bioactive glasses with the HPTS was investigated by steady-state luminescence and excited-state lifetime measurements. The prepared composites showed linearizable responses when exposed to different CO2 concentrations. High I0/I100 values (87, 109 and 115) were obtained for the binary blends of HPTS and bioactive glasses. When the imidazolium-based IL was used together with the offered composites, we observed improved stability and longevity for the HPTS up to 315 days. The incorporation of imidazolium-based ionic liquids as additives in HPTS-based fluorescence assays together with the bioactive glasses holds promise for enhancing the photostability of the HPTS and improving the reliability and longevity of fluorescence signals in various research applications.

DNA polymerase ζ is a robust reverse transcriptase.

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway.

AKT-mediated phosphorylation of TSC2 controls stimulus- and tissue-specific mTORC1 signaling and organ growth.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

GRAIL1 Stabilizes Misfolded Mutant p53 through a Ubiquitin Ligase-Independent, Chaperone Regulatory Function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's esophagus progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL-gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But, how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here, we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for heat shock protein 40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain or a cell-permeable peptide (Pep-J) encoding the above 10 amino acids can bind and inhibit DNAJ-Hsp70 co-chaperone activity, thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic Barrett's esophagus and EAC cells and inhibit the growth of patient-derived organoids of dysplastic Barrett's esophagus in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J are comparable with simvastatin, a cholesterol-lowering drug that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase-independent, chaperone-regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

Primary cell cultures from the single-chromosome ant Myrmecia croslandi.

The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis.

The Caenorhabditis elegans cuticle and precuticle: a model for studying dynamic apical extracellular matrices in vivo.

Apical extracellular matrices (aECMs) coat the exposed surfaces of animal bodies to shape tissues, influence social interactions, and protect against pathogens and other environmental challenges. In the nematode Caenorhabditis elegans, collagenous cuticle and zona pellucida protein-rich precuticle aECMs alternately coat external epithelia across the molt cycle and play many important roles in the worm's development, behavior, and physiology. Both these types of aECMs contain many matrix proteins related to those in vertebrates, as well as some that are nematode-specific. Extensive differences observed among tissues and life stages demonstrate that aECMs are a major feature of epithelial cell identity. In addition to forming discrete layers, some cuticle components assemble into complex substructures such as ridges, furrows, and nanoscale pillars. The epidermis and cuticle are mechanically linked, allowing the epidermis to sense cuticle damage and induce protective innate immune and stress responses. The C. elegans model, with its optical transparency, facilitates the study of aECM cell biology and structure/function relationships and all the myriad ways by which aECM can influence an organism.

Proteomics Analysis of Interactions between Drug-Resistant and Drug-Sensitive Cancer Cells: Comparative Studies of Monoculture and Coculture Cell Systems.

Cell-cell interactions, which allow cells to communicate with each other through molecules in their microenvironment, are critical for the growth, health, and functions of cells. Previous studies show that drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level, which is partially responsible for cancer recurrence. Studying protein targets and pathways involved in cell-cell communication provides essential information for fundamental cell biology studies and therapeutics of human diseases. In the current studies, we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication. Comparative studies were carried out using monoculture cells. Shotgun bottom-up proteomics results indicate that the P53 signaling pathway has a strong association with drug resistance mechanisms, and multiple TP53-related proteins were upregulated in both direct and indirect coculture systems. In addition, cell-cell communication pathways, including the phagosome and the HIF-signaling pathway, contribute to both direct and indirect coculture systems. Consequently, AK3 and H3-3A proteins were identified as potential targets for cell-cell interactions that are relevant to drug resistance mechanisms. We propose that the P53 signaling pathway, in which mitochondrial proteins play an important role, is responsible for inducing drug resistance through communication between drug-resistant and drug-sensitive cancer cells.

High-Q WGM microcavity-based optofluidic sensor technologies for biological analysis.

High-quality-factor (Q) optical microcavities have attracted extensive interest due to their unique ability to confine light for resonant circulation at the micrometer scale. Particular attention has been paid to optical whispering-gallery mode (WGM) microcavities to harness their strong light-matter interactions for biological applications. Remarkably, the combination of high-Q optical WGM microcavities with microfluidic technologies can achieve a synergistic effect in the development of high-sensitivity optofluidic sensors for many emerging biological analysis applications, such as the detection of proteins, nucleic acids, viruses, and exosomes. They can also be utilized to investigate the behavior of living cells in human organisms, which may provide new technical solutions for studies in cell biology and biophysics. In this paper, we briefly review recent progress in high-Q microcavity-based optofluidic sensor technologies and their applications in biological analysis.

Bisphenol A (BPA) and neurological disorders: An overview

The human body is commonly exposed to bisphenol A (BPA), which is widely used in consumer and industrial products. BPA is an endocrine-disrupting chemical that has adverse effects on human health. In particular, many studies have shown that BPA can cause various neurological disorders by affecting brain development and neural function during prenatal, infancy, childhood, and adulthood exposure. In this review, we discussed the correlation between BPA and neurological disorders based on molecular cell biology, neurophysiology, and behavioral studies of the effects of BPA on brain development and function. Recent studies, both animal and epidemiological, strongly indicate that BPA significantly impacts brain development and function. It hinders neural processes, such as proliferation, migration, and differentiation during development, affecting synaptic formation and activity. As a result, BPA is implicated in neurodevelopmental and neuropsychiatric disorders like autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD), and schizophrenia.

Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Abstract PO-012: Gain-of-function small molecules to reprogram oncogenic transcription in lymphoma

Abstract Conventional pharmacotherapy in oncology centers on target-centric silencing of aberrant protein function through small molecule inhibitors, degraders, or CRISPR/RNAi. In contrast, here we leverage induced proximity to develop pharmacology that activates dominant signaling pathways for a therapeutic effect. To develop this concept, we describe a new class of bivalent small molecules that reprogram cancer-driving gene expression to activate cell death signaling in diffuse large B cell lymphomas (DLBCL). These molecules, called Transcriptional Chemical Inducers of Proximity (TCIPs), operate at fractional occupancy of their protein targets, without genomic modifications to the cell or organism. The first TCIPs transiently re-localized elongation factors such as BRD4 (TCIP1, PMID 37495688) to loci regulated by the transcription factor BCL6, inducing the expression of pro-apoptotic genes regulated by BCL6. In new, unpublished work, CDK-TCIPs have been made to re-direct the activity of transcriptional elongation kinases such as CDK9, CDK12, or CDK13 to induce the expression of pro-apoptotic BCL6-target genes. Profiling in ∼900 barcoded cancer cell lines show that the molecules exhibit potent cell killing potency in a highly DLBCL lineage-restricted manner. Cytotoxicity to healthy lymphocytes is 100- to 10,000-fold lower, although germinal center B cells are ablated in a dose-dependent manner consistent with the role of BCL6 in the germinal center. Biochemical, cell biological, proteomic, and genomic studies demonstrate that CDK-TCIPs operate via a gain-of-function mechanism reliant on the ternary complex formed by the bivalent molecule. Instead of global enzyme inhibition or degradation, CDK-TCIPs borrow and re-localize a fraction of total CDK activity to BCL6-bound chromatin, producing pharmacology from the induction of pro-apoptotic gene transcription. The findings suggest a path to rational design of new gain-of-function chemotherapeutic strategies to rewire cell signaling using induced proximity. Citation Format: Sai Gourisankar, Roman Sarott, Basel Karim, Sabin Nettles, Haopeng Yang, Brendan Dwyer, Juste Simanauskaite, Jason Tse, Hind Abuzaid, Stephen M Hinshaw, Michael R Green, Gerald R Crabtree, Nathanael S Gray. Gain-of-function small molecules to reprogram oncogenic transcription in lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-012.

Dual-Stimuli Regulation of DNAzyme Cleavage Reaction by Coordination-Driven Nanoprobes for Cancer Cell Imaging.

Endowing current artificial chemical reactions (ACRs) with high specificity and intricate activation capabilities is crucial for expanding their applications in accurate bioimaging within living cells. However, most of the reported ACR-based evaluations relied on either single biomarker stimuli or dual activators without obvious biological relevance, still limiting their accuracy and fidelity. Herein, taking the metal-ion-dependent DNAzyme cleavage reaction as a model ACR, two regulators, glutathione (GSH) and telomerase (TE) activated DNAzyme cleavage reactions, were exploited for precise discrimination of cancerous cells from normal cells. DNA probe was self-assembled into the ZIF-90 nanoparticle framework to construct coordination-driven nanoprobes. This approach enhances the stability and specificity of tumor imaging by utilizing biomarkers associated with rapid tumor proliferation and those commonly overexpressed in tumors. In conclusion, the research not only paves the way for new perspectives in cell biology and pathology studies but also lays a solid foundation for the advancement of biomedical imaging and disease diagnostic technologies.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.