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Sorted publications based on keyword
Gurtner, A;Borrelli, C;Gonzalez-Perez, I;Bach, K;Acar, IE;Núñez, NG;Crepaz, D;Handler, K;Vu, VP;Lafzi, A;Stirm, K;Raju, D;Gschwend, J;Basler, K;Schneider, C;Slack, E;Valenta, T;Becher, B;Krebs, P;Moor, AE;Arnold, IC;
Nature,
615,
(7950),
151-157,
(2023)
In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.
de Ceglia, R;Ledonne, A;Litvin, DG;Lind, BL;Carriero, G;Latagliata, EC;Bindocci, E;Di Castro, MA;Savtchouk, I;Vitali, I;Ranjak, A;Congiu, M;Canonica, T;Wisden, W;Harris, K;Mameli, M;Mercuri, N;Telley, L;Volterra, A;
Nature,
622,
(7981),
120-129,
(2023)
Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.
Goh, I;Botting, RA;Rose, A;Webb, S;Engelbert, J;Gitton, Y;Stephenson, E;Quiroga Londoño, M;Mather, M;Mende, N;Imaz-Rosshandler, I;Yang, L;Horsfall, D;Basurto-Lozada, D;Chipampe, NJ;Rook, V;Lee, JTH;Ton, ML;Keitley, D;Mazin, P;Vijayabaskar, MS;Hannah, R;Gambardella, L;Green, K;Ballereau, S;Inoue, M;Tuck, E;Lorenzi, V;Kwakwa, K;Alsinet, C;Olabi, B;Miah, M;Admane, C;Popescu, DM;Acres, M;Dixon, D;Ness, T;Coulthard, R;Lisgo, S;Henderson, DJ;Dann, E;Suo, C;Kinston, SJ;Park, JE;Polanski, K;Marioni, J;van Dongen, S;Meyer, KB;de Bruijn, M;Palis, J;Behjati, S;Laurenti, E;Wilson, NK;Vento-Tormo, R;Chédotal, A;Bayraktar, O;Roberts, I;Jardine, L;Göttgens, B;Teichmann, SA;Haniffa, M;
Science (New York, N.Y.),
381,
(6659),
eadd7564,
(2023)
The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.
Li, S;Kong, L;Meng, Y;Cheng, C;Lemacon, DS;Yang, Z;Tan, K;Cheruiyot, A;Lu, Z;You, Z;
Molecular cell,
83,
(4),
556-573.e7,
(2023)
The protection of DNA replication forks under stress is essential for genome maintenance and cancer suppression. One mechanism of fork protection involves an elevation in intracellular Ca2+ ([Ca2+]i), which in turn activates CaMKK2 and AMPK to prevent uncontrolled fork processing by Exo1. How replication stress triggers [Ca2+]i elevation is unclear. Here, we report a role of cytosolic self-DNA (cytosDNA) and the ion channel TRPV2 in [Ca2+]i induction and fork protection. Replication stress leads to the generation of ssDNA and dsDNA species that, upon translocation into cytoplasm, trigger the activation of the sensor protein cGAS and the production of cGAMP. The subsequent binding of cGAMP to STING causes its dissociation from TRPV2, leading to TRPV2 derepression and Ca2+ release from the ER, which in turn activates the downstream signaling cascade to prevent fork degradation. This Ca2+-dependent genome protection pathway is also activated in response to replication stress caused by oncogene activation.
Ast, J;Nasteska, D;Fine, NHF;Nieves, DJ;Koszegi, Z;Lanoiselée, Y;Cuozzo, F;Viloria, K;Bacon, A;Luu, NT;Newsome, PN;Calebiro, D;Owen, DM;Broichhagen, J;Hodson, DJ;
Nature communications,
14,
(1),
301,
(2023)
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.
Iwasaki, M;Lefevre, A;Althammer, F;Clauss Creusot, E;Łąpieś, O;Petitjean, H;Hilfiger, L;Kerspern, D;Melchior, M;Küppers, S;Krabichler, Q;Patwell, R;Kania, A;Gruber, T;Kirchner, MK;Wimmer, M;Fröhlich, H;Dötsch, L;Schimmer, J;Herpertz, SC;Ditzen, B;Schaaf, CP;Schönig, K;Bartsch, D;Gugula, A;Trenk, A;Blasiak, A;Stern, JE;Darbon, P;Grinevich, V;Charlet, A;
Nature communications,
14,
(1),
1066,
(2023)
The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.
Massier, L;Jalkanen, J;Elmastas, M;Zhong, J;Wang, T;Nono Nankam, PA;Frendo-Cumbo, S;Bäckdahl, J;Subramanian, N;Sekine, T;Kerr, AG;Tseng, BTP;Laurencikiene, J;Buggert, M;Lourda, M;Kublickiene, K;Bhalla, N;Andersson, A;Valsesia, A;Astrup, A;Blaak, EE;Ståhl, PL;Viguerie, N;Langin, D;Wolfrum, C;Blüher, M;Rydén, M;Mejhert, N;
Nature communications,
14,
(1),
1438,
(2023)
To date, single-cell studies of human white adipose tissue (WAT) have been based on small cohort sizes and no cellular consensus nomenclature exists. Herein, we performed a comprehensive meta-analysis of publicly available and newly generated single-cell, single-nucleus, and spatial transcriptomic results from human subcutaneous, omental, and perivascular WAT. Our high-resolution map is built on data from ten studies and allowed us to robustly identify >60 subpopulations of adipocytes, fibroblast and adipogenic progenitors, vascular, and immune cells. Using these results, we deconvolved spatial and bulk transcriptomic data from nine additional cohorts to provide spatial and clinical dimensions to the map. This identified cell-cell interactions as well as relationships between specific cell subtypes and insulin resistance, dyslipidemia, adipocyte volume, and lipolysis upon long-term weight changes. Altogether, our meta-map provides a rich resource defining the cellular and microarchitectural landscape of human WAT and describes the associations between specific cell types and metabolic states.
Manzato, C;Larini, L;Pegorar, CO;Dello Stritto, MR;Jurikova, K;Jantsch, V;Cusanelli, E;
Nucleic acids research,
(2023)
Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific mechanisms regulate its biogenesis and function. Here we report on the expression of TERRA in Caenorhabditis elegans. We show that C. elegans TERRA is regulated by the telomere-binding proteins POT-1 and POT-2 which repress TERRA in a telomere-specific manner. C. elegans TERRA transcripts are heterogeneous in length and form discrete nuclear foci, as detected by RNA FISH, in both postmitotic and germline cells; a fraction of TERRA foci localizes to telomeres. Interestingly, in germ cells, TERRA is expressed in all stages of meiotic prophase I, and it increases during pachytene, a stage in meiosis when homologous recombination is ongoing. We used the MS2-GFP system to study the spatiotemporal dynamics of single-telomere TERRA molecules. Single particle tracking revealed different types of motilities, suggesting complex dynamics of TERRA transcripts. Finally, we unveiled distinctive features of C. elegans TERRA, which is regulated by telomere shortening in a telomere-specific manner, and it is upregulated in the telomerase-deficient trt-1; pot-2 double mutant prior to activation of the alternative lengthening mechanism ALT. Interestingly, in these worms TERRA displays distinct dynamics with a higher fraction of fast-moving particles.
Lago, C;Gianesello, M;Santomaso, L;Leva, G;Ballabio, C;Anderle, M;Antonica, F;Tiberi, L;
Nature protocols,
(2023)
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Goodwin, K;Lemma, B;Zhang, P;Boukind, A;Nelson, CM;
Developmental cell,
58,
(5),
338-347.e4,
(2023)
It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.
Price, KL;Tharakan, DM;Cooley, L;
Developmental cell,
58,
(6),
474-488.e5,
(2023)
How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called “ring canals,” is poorly understood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. Our results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.
Pellegrini, FR;De Martino, S;Fianco, G;Ventura, I;Valente, D;Fiore, M;Trisciuoglio, D;Degrassi, F;
Autophagy,
1-16,
(2023)
Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.
Yang, W;Hou, L;Luo, C;
Small (Weinheim an der Bergstrasse, Germany),
19,
(23),
e2207341,
(2023)
Super-resolution microscopy is rapidly developed in recent years, allowing biologists to extract more quantitative information on subcellular processes in live cells that is usually not accessible with conventional techniques. However, super-resolution imaging is not fully exploited because of the lack of an appropriate and multifunctional experimental platform. As an important tool in life sciences, microfluidics is capable of cell manipulation and the regulation of the cellular environment because of its superior flexibility and biocompatibility. The combination of microfluidics and super-resolution microscopy revolutionizes the study of complex cellular properties and dynamics, providing valuable insights into cellular structure and biological functions at the single-molecule level. In this perspective, an overview of the main advantages of microfluidic technology that are essential to the performance of super-resolution microscopy are offered. The main benefits of performing super-resolution imaging with microfluidic devices are highlighted and perspectives on the diverse applications that are facilitated by combining these two powerful techniques are provided.
García-Revilla, J;Boza-Serrano, A;Jin, Y;Vadukul, DM;Soldán-Hidalgo, J;Camprubí-Ferrer, L;García-Cruzado, M;Martinsson, I;Klementieva, O;Ruiz, R;Aprile, FA;Deierborg, T;Venero, JL;
Acta neuropathologica,
146,
(1),
51-75,
(2023)
Parkinson’s Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identified an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fibrils. In addition, aggregation experiments show that Gal3 affects spatial propagation and the stability of pre-formed αSyn fibrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD.
Di Sante, M;Antonucci, S;Pontarollo, L;Cappellaro, I;Segat, F;Deshwal, S;Greotti, E;Grilo, LF;Menabò, R;Di Lisa, F;Kaludercic, N;
Basic research in cardiology,
118,
(1),
4,
(2023)
During embryonic development, cardiomyocytes undergo differentiation and maturation, processes that are tightly regulated by tissue-specific signaling cascades. Although redox signaling pathways involved in cardiomyogenesis are established, the exact sources responsible for reactive oxygen species (ROS) formation remain elusive. The present study investigates whether ROS produced by the mitochondrial flavoenzyme monoamine oxidase A (MAO-A) play a role in cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs). Wild type (WT) and MAO-A knock out (KO) hiPSCs were generated by CRISPR/Cas9 genome editing and subjected to cardiomyocyte differentiation. Mitochondrial ROS levels were lower in MAO-A KO compared to the WT cells throughout the differentiation process. MAO-A KO hiPSC-derived cardiomyocytes (hiPSC-CMs) displayed sarcomere disarray, reduced α- to β-myosin heavy chain ratio, GATA4 upregulation and lower macroautophagy levels. Functionally, genetic ablation of MAO-A negatively affected intracellular Ca2+ homeostasis in hiPSC-CMs. Mechanistically, MAO-A generated ROS contributed to the activation of AKT signaling that was considerably attenuated in KO cells. In addition, MAO-A ablation caused a reduction in WNT pathway gene expression consistent with its reported stimulation by ROS. As a result of WNT downregulation, expression of MESP1 and NKX2.5 was significantly decreased in MAO-A KO cells. Finally, MAO-A re-expression during differentiation rescued expression levels of cardiac transcription factors, contractile structure, and intracellular Ca2+ homeostasis. Taken together, these results suggest that MAO-A mediated ROS generation is necessary for the activation of AKT and WNT signaling pathways during cardiac lineage commitment and for the differentiation of fully functional human cardiomyocytes.
Jacob, T;Annusver, K;Czarnewski, P;Dalessandri, T;Kalk, C;Levra Levron, C;Campamà Sanz, N;Kastriti, ME;Mikkola, ML;Rendl, M;Lichtenberger, BM;Donati, G;Björklund, ÅK;Kasper, M;
Developmental cell,
(2023)
A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.
Ju, Z;Lei, M;Xuan, L;Luo, J;Zhou, M;Wang, Y;Shen, L;Skonieczna, M;Ivanov, DS;Zakaly, HMH;Markovic, V;Zhou, P;Huang, R;
Journal of advanced research,
(2023)
p53 wild-type lung cancer cells can develop radiation resistance. Circular RNA (circRNA) consists of a family of transcripts with exclusive structures. circRNA is critical in tumorigenesis and is a potential biomarker or therapeutic target. It is uncertain how circRNA expression and functions are regulated post-radiation in p53 wild-type cancer cells.A549 or H1299 cells were divided into p53-wt and p53-KO groups by CRISPR/Cas9; both groups were subjected to 4 Gy ionizing radiation (IR: p53-wt-IR and p53-KO-IR). RNA-seq, CCK8, cell cycle, and other functional and mechanism experiments were performed in vivo. p53 gene knockout mice were generated to test the cell results in vitro.circRNAs were found in differential groups. circRNA_0006420 (IRSense) was upregulated in p53-wt cells but had the same expression level as p53-KO cells after radiation, indicating that p53 silencing prevents its upregulation after IR. In the presence of p53, upregulated IRSense post-radiation induces G2/M arrest by regulating DNA damage repair (DDR) pathway-related proteins. Meanwhile, upregulated IRSense post-radiation aggravates the radiation-induced epithelial-mesenchymal transition (EMT). Interestingly, in the presence of p53, it promotes IRSense/HUR/PTBP1 complex formation resulting in the promotion of the radiation-induced EMT. Moreover, c-Jun regulates the upregulation of p53 transcription after radiation treatment. For these lung cancer cells with p53, upregulated IRSense aggravates lung cancer cell proliferation and increases radiation resistance by interacting with HUR (ElAV-like protein 1) and PTBP1 (polypyrimidine tract-binding protein 1) in the nucleus.Lung cancer cells retaining p53 may upregulate circRNA_0006420 (IRSense) expression post radiation to form an IRSense/HUR/PTBP1 complex leading to radiotherapy resistance. This study furthers our understanding of the roles of circRNA in regulating the effect of radiotherapy and provides novel therapeutic avenues for effective clinical lung cancer therapies.
Mallick, S;Chakrabarti, J;Eschbacher, J;Moraitis, AG;Greenstein, AE;Churko, J;Pond, KW;Livolsi, A;Thorne, CA;Little, AS;Yuen, KCJ;Zavros, Y;
Translational research : the journal of laboratory and clinical medicine,
(2023)
Cushing’s disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that that subsequently leads to chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced a predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of Cushing’s disease patients.
Zhang, Y;Rózsa, M;Liang, Y;Bushey, D;Wei, Z;Zheng, J;Reep, D;Broussard, G;Tsang, A;Tsegaye, G;Narayan, S;Obara, C;Lim, J;Patel, R;Zhang, R;Ahrens, M;Turner, G;Wang, S;Korff, W;Schreiter, E;Svoboda, K;Hasseman, J;Kolb, I;Looger, L.
Nature,
615,
884–891,
(2023)
Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems. The popular GCaMP indicators are based on the calcium-binding protein calmodulin and the RS20 peptide. These sensors report neural activity at timescales much slower than electrical signaling, limited by their biophysical properties and trade-offs between sensitivity and speed. We used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting ‘jGCaMP8’ sensors, based on calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (rise times, 2 ms) and still feature the highest sensitivity for neural activity reported for any protein-based sensor. jGCaMP8 sensors will allow tracking of larger populations of neurons on timescales relevant to neural computation.
Van der Meeren, L;Efimova, I;Demuynck, R;Parakhonskiy, B;Krysko, DV;Skirtach, AG;
Advanced healthcare materials,
e2301025,
(2023)
The importance of the clearance of dead cells is shown to have a regulatory role for normal tissue homeostasis and for the modulation of immune responses. However, how mechanobiological properties of dead cells affect efferocytosis remains largely unknown. Here, it is reported that the Young’s modulus of cancer cells undergoing ferroptosis is reduced. To modulate their Young’s modulus a layer-by-layer (LbL) nanocoating is developed. Scanning electron and fluorescence microscopy confirm coating efficiency of ferroptotic cells while atomic force microscopy reveals encapsulation of the dead cells increases their Young’s modulus dependent on the number of applied LbL layers which increases their efferocytosis by primary macrophages. This work demonstrates the crucial role of mechanobiology of dead cells in regulating their efferocytosis by macrophages which can be exploited for the development of novel therapeutic strategies for diseases where modulation of efferocytosis can be potentially beneficial and for the design of drug delivery systems for cancer therapy.

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