Culture Model Research Articles

SHOC2 plays an oncogenic or tumor suppressive role by differentially targeting the MAPK and mTORC1 signals in liver cancer

Abstract SHOC2 is a scaffold protein that activates the RAS-MAPK signal. Our recent study showed that SHOC2 is also a negative regulator of the mTORC1 signal in lung cancer cells. Whether and how SHOC2 differentially regulates the RAS-MAPK vs. the mTORC1 signals in liver cancer remains unknown. Here we showed that SHOC2 is overexpressed in human liver cancer tissues, and SHOC2 overexpression promotes growth and survival of liver cancer cells via activation of the RAS-MAPK signal, although the mTORC1 signal is inactivated. SHOC2 knockdown suppresses the growth of liver cancer cells mainly through inactivating the RAS-MAPK signal. Thus, in the cell culture models, SHOC2 regulation of growth is dependent of the RAS-MAPK, but not the mTORC1 signal. Interestingly, in a mouse liver cancer model induced by diethylnitrosamine (DEN)-high fat diet (HFD), hepatocyte-specific Shoc2 deletion inactivates the Ras-Mapk signal, but has no effect in liver tumorigenesis. However, in the Pten loss-induced liver cancer model, Shoc2 deletion further activates mTorc1 without affecting the Ras-Mapk signal, and promotes liver tumorigenesis. Collectively, it appears that SHOC2 could act as either an oncogene (via activating the MAPK signal) or a tumor suppressor (via inactivating the mTORC1 signal) in the manner dependent of the dominancy of the MAPK vs. mTORC1 signals.

Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes.

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

Sulforaphane and ophthalmic diseases

AbstractSulforaphane (SFN) is an organosulfur compound categorized as an isothiocyanate (ITC), primarily extracted from cruciferous vegetables like broccoli and cabbage. The molecular formula of sulforaphane (SFN) is C6H11NOS2. SFN is generated by the hydrolysis of glucoraphanin (GRP) through the enzyme myrosinase, showing notable properties including anti‐diabetic, anti‐inflammatory, antimicrobial, anti‐angiogenic, and anticancer attributes. Ongoing clinical trials are investigating its potential in diseases such as cancer, neurodegenerative diseases, diabetes‐related complications, chronic kidney disease, cardiovascular disease, and liver diseases. Several animal carcinogenesis models and cell culture models have shown it to be a very effective chemopreventive agent, and the protective effects of SFN in ophthalmic diseases have been linked to multiple mechanisms. In murine models of diabetic retinopathy and age‐related macular degeneration, SFN delays retinal photoreceptor cell degeneration through the Nrf2 antioxidative pathway, NF‐κB pathway, AMPK pathway, and Txnip/mTOR pathway. In rabbit models of keratoconus and cataract, SFN has been shown to protect corneal and lens epithelial cells from oxidative stress injury by activating the Keap1‐Nrf2‐ARE pathway and the Nrf‐2/HO‐1 antioxidant pathway. Oral delivery or intraperitoneal injection at varying concentrations are the primary strategies for SFN intake in current preclinical studies. Challenges remain in the application of SFN in eye disorders due to its weak solubility in water and limited bioavailability because of the presence of blood–ocular barrier systems. This review comprehensively outlines recent research on SFN, elucidates its mechanisms of action, and discusses potential therapeutic benefits for eye disorders such as age‐related macular degeneration (AMD), diabetic retinopathy (DR), cataracts, and other ophthalmic diseases, while also indicating directions for future clinical research to achieve efficient SFN treatment for ophthalmic diseases.

Tracking Drugs and Lipids: Quantitative Mass Spectrometry Imaging of Liposomal Doxorubicin Delivery and Bilayer Fate in Three-Dimensional Tumor Models.

Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.

GLUT2 regulation of p38 MAPK isoform protein expression and p38 phosphorylation in male versus female rat hypothalamic primary astrocyte Cultures

Recent studies documented regulation of hypothalamic astrocyte mitogen-activated protein kinase (MAPK) pathways, including p38, by the plasma membrane glucose carrier/sensor glucose transporter-2 (GLUT2). Sex-specific GLUT2 control of p38 phosphorylation was observed, but effects on individual p38 family protein profiles were not investigated. Current research employed an established primary astrocyte culture model, gene knockdown tools, and selective primary antisera against p38-alpha, p38-beta, p38-gamma, and p38-delta isoforms to investigate whether GLUT2 governs expression of one or more of these variants in a glucose-dependent manner. Data show that GLUT2 inhibits baseline expression of each p38 protein in male cultures, yet stimulates p38-delta profiles without affecting other p38 proteins in female. Glucose starvation caused selective up-regulation of p38-delta profiles in male versus p38-alpha and -gamma proteins in female; these positive responses were amplified by GLUT2 siRNA pretreatment. GLUT2 opposes or enhances basal p38 phosphorylation in male versus female, respectively. GLUT2 siRNA pretreatment did not affect glucoprivic patterns of phospho-p38 protein expression in either sex. Outcomes document co-expression of the four principal p38 MAPK family proteins in hypothalamic astrocytes, and implicate GLUT2 in regulation of all (male) versus one (female) variant(s). Glucoprivation up-regulated expression of distinctive p38 isoforms in each sex; these stimulatory responses are evidently blunted by GLUT2. Glucoprivic-associated loss of GLUT2 gene silencing effects on p38 phosphorylation infers either that glucose status determines whether this sensor controls phosphorylation, or that decrements in screened glucose in each instance are of sufficient magnitude to abolish GLUT2 regulation of that function.

The influence of viscosity of hydrogels on the spreading and migration of cells in 3D bioprinted skin cancer models.

Various in vitro three-dimensional (3D) tissue culture models of human and diseased skin exist. Nevertheless, there is still room for the development and improvement of 3D bioprinted skin cancer models. The need for reproducible bioprinting methods, cell samples, biomaterial inks, and bioinks is becoming increasingly important. The influence of the viscosity of hydrogels on the spreading and migration of most types of cancer cells is well studied. There are however limited studies on the influence of viscosity on the spreading and migration of cells in 3D bioprinted skin cancer models. In this review, we will outline the importance of studying the various types of skin cancers by using 3D cell culture models. We will provide an overview of the advantages and disadvantages of the various 3D bioprinting technologies. We will emphasize how the viscosity of hydrogels relates to the spreading and migration of cancer cells. Lastly, we will give an overview of the specific studies on cell migration and spreading in 3D bioprinted skin cancer models.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Anti-neurofascin-155 antibody mediated a distinct phenotype of chronic inflammatory demyelinating polyradiculoneuropathy.

To investigate Ranvier's autoantibodies prevalence and isotypes in various peripheral neuropathy variants, compare clinical features between seronegative and seropositive patients, and elucidate immune mechanisms underlying antibody generation. Antibodies against anti-neurofascin-155 (NF155), NF186, contactin-1 (CNTN1), CNTN2, contactin-associated protein 1 (CASPR1), and CASPR2 were identified through cell-based assays. Plasma cytokines were analyzed in anti-NF155 antibody-positive chronic inflammatory demyelinating polyneuropathy (NF155+ CIDP) and Ranvier's antibodies-negative CIDP (Ab- CIDP) patients using a multiplexed fluorescent immunoassay, validated in vitro in a cell culture model. In 368 plasma samples, 50 Ranvier's autoantibodies were found in 45 individuals, primarily in CIDP cases (25 out of 69 patients) and in 10 out of 122 Guillain-Barré syndrome patients. Anti-NF155 and CNTN1-IgG were exclusive to CIDP. Fourteen samples were NF155-IgG, primarily IgG4 subclass, linked to CIDP features including early onset, tremor, sensory disturbance, elevated CSF protein, prolonged motor latency, conduction block, and poor treatment response. NF155-IgG had low sensitivity (20.28%) but high specificity (100%) for CIDP, rising to 88.88% with tremor and prolonged motor latency. Cytokine profiling in NF155+ CIDP revealed distinct immune responses involving helper T cells, toll-like receptor pathways. Some NF155+ CIDP patients had circulating NF155-specific B cells producing NF155-IgG without antigen presence, suggesting therapeutic potential. The study emphasizes the high specificity and sensitivity of NF155-IgG for diagnosing CIDP characterized by distinctive features. Further investigation into circulating NF155-specific B cell phenotypes may pave the way for B cell directed therapy.

A 'Cultural Models' Approach to Psychotherapy for Refugees and Asylum Seekers: A Case Study from the UK.

Despite the existence of significant research on the mental health care challenges of migrants, particularly refugees and asylum seekers, less attention has been paid to treatment approaches. We used a case study from the UK to look at the topic from a cultural models approach (which comes from cognitive anthropology) to analyse migrants' experiences with mental health care. Twenty-five refugees and asylum seekers living in North East England and Northern Ireland were interviewed who had used at least six sessions of talking therapy during the last three years. Our results suggested that adopting a 'cultural models' approach, which offers a new conceptual and methodological framework of migrants' experiences and their underlying schemas and expectations, would significantly contribute to building therapeutic alliances and provide relevant and appropriate treatments for migrant clients, particularly for unrecognised pre- and post-migration traumatic experiences.

Exploring EFL learners’ actions during the use of lecture materials in Chinese tertiary classrooms from a transactional view

Learners’ interaction with lecture materials has remained underexplored despite the growing body of studies on the use of materials. The current study explored the actions EFL learners employed during the use of lecture materials in tertiary classrooms in China through the lens of a transactional view. This empirical study involved forty-two students in two classes as well as two instructors enrolled in the Comprehensive English Course. Qualitative analysis revealed five levels of learner actions during interactions initiated by lectures: awareness, involvement, intervention, creation, and autonomy. The study further highlighted three factors that significantly influence these actions: learners’ intrinsic motivation to participate in classroom interactions, discourse space for participation, and the cultural model of learning. Pedagogical implications for enhancing learner autonomy and contributions during lecture materials use were discussed based on results.

Addressing Challenges and Promoting Development: Strategies for Health and Wellness Industry in Ethnic Regions

China has entered an aging society. In order to respond to the aging society, government departments have successively introduced various pension policies since 2013 with an attempt to mitigate the impact of the aging society. Under this context, the health and wellness industry came into being. However, the documents of the central department for the health and wellness industry are still lacking, the health care issues in ethnic region have received less attention, and there is no specific legal source for local governments and enterprises to follow. The purpose of this paper is to clarify the development of the health and wellness industry in ethnic region through literature analysis, to study the feasible path of the development of the health and wellness industry in ethnic region, and to provide suggestions. This study finds 40 representative papers on rural revitalization and health and wellness industry in the past three years through literature retrieval. We summarize the questions raised in the papers, analyzes and evaluates them, and finally proposes a conclusion. Findings show that the development of health and wellness industry in ethnic region lacks national level policies, and the details of local implementation are relatively weak. Moreover, in terms of the development path of the health and wellness industry in ethnic region, the integration model of health care, sports, culture and tourism seems to be a feasible direction. In addition, the development of health and wellness industry in ethnic region has a positive impact on rural revitalization. In other words, the development of health and wellness industry in ethnic region may have a high coupling effect with regional economy. Finally, this study provides some useful suggestions for government departments and subsequent research.

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

P04 How fibroblasts and azathioprine prepare the soil for cutaneous squamous cell carcinoma

Abstract Introduction and aims DNA damage-inducing immunosuppressive drugs such as azathioprine (AZA) act synergistically with ultraviolet (UV)A radiation and photosensitize the skin of immunosuppressed individuals, such as organ transplant recipients. These individuals have an increased risk of developing cutaneous squamous cell carcinoma (cSCC). The specific combination of AZA and UVA causes oxidative DNA damage that leaves behind a characteristic mutational signature in tumour keratinocytes of patients treated with AZA. However, as UVA radiation can penetrate the dermis, the impact of AZA/UVA on cells of the tumour microenvironment, such as fibroblasts, also need to be addressed. Fibroblasts are crucial for cSCC development and progression as they can switch to a protumourigenic phenotype. Methods Cell viability and senescence assays were performed to investigate the impact of AZA in combination with or without UVA on neonatal (nonsun-exposed) and adult (sun-exposed) human dermal fibroblasts. In addition, the impact of AZA/UVA-treated fibroblasts on the clonal growth properties of different cSCC cell models was investigated using three-dimensional (co)culture models. Results We found that AZA and UVA synergistically reduce the viability of both UV-naïve neonatal and sun-exposed adult dermal fibroblasts. In addition, combined AZA and UVA treatments lead to an increased percentage of senescent fibroblasts. We hypothesize that the aberrant secretome of these senescent fibroblasts can impact the growth behaviour of tumour cells. In a soft agar assay, the more malignant cSCC cells (MET-1) formed more and larger clonal spheroids and were less dependent on growth factors compared with spheroids formed by premalignant (PM-1) keratinocytes. Conclusions AZA and UVA act synergistically and potentiate a UVA-induced senescent fibroblast phenotype. The changed characteristics of these fibroblasts can impact the stem cell-like properties from premalignant and malignant keratinocytes. Combining keratinocyte and fibroblast culture models enables us to study how AZA/UVA promote the initiation and progression of cSCC.

The Mode of Being of Popular Literature in the 20th Century

The article is devoted to the functioning of literature (and, more broadly, cultural texts) in popular circulation and the transformation of their role in the 20th century. The starting point for the analysis is the link between commercialisation (as an immanent feature of the ‘lowbrow’ circulation) and the artistic quality of the output that is part of it. The examples of the phenomena under discussion come from different linguistic milieux. This highlights the transnational nature of popular works, which feature invariant solutions to plots. In these two contradictory yet simultaneous tendencies can be observed: schematisation and de-schematisation, which renews conventionalised solutions. The author also emphasises the role of English-language works in the creation of figures of the collective imagination. Their presence in the minds of readers and the transformations they undergo with the development of industrial society and urbanisation processes can be traced from the late 19th century to the 1990s. That is why the 20th century — a period of intense expansion of popular culture — is a cohesive cultural entity with distinguishable individual periods. They are usually associated with socio-political crises, with stories becoming a cultural response to them and, at the same time, their artistic reception in the imaginarium communis. In addition, this is a time of technological progress, significantly affecting the distribution and media-based mediation of cultural texts in popular circulation. Literature has ceased to be a ‘separate phenomenon’ in it, hence the need to look at it as a part of a larger whole, with which it enters into various relationships. The domination of English-language output during the analysed period is associated with the global hegemony of the Anglo-American entertainment model. At the same time — in the works available to Polish-speaking readers — its ‘over-presence’ stems from the appearance after 1989 of a large number of translations, which have significantly influenced domestic pop-culture; moreover, their popularity in the reality of the free market economy has been determined by the read- ers themselves with their purchasing choices. It is hard to speak in this situation of the existence of national models of popular culture, although undeniably there are authors who dominate the local publishing markets. And yet they, too, exist in the context of the global pop culture industry.

Cultural Competence Interventions in European Healthcare: A Scoping Review.

Europe is undergoing rapid social change and is distinguished by its cultural superdiversity. Healthcare is facing an increasing need for professionals to adapt to this environment. Thus, the promotion of cultural competence in healthcare has become a priority. However, the training being developed and their suitability for the European context are not well known. The aim of this qualitative study has been to map the scientific literature in order to comprehend the current state of research on this topic. For this purpose, we conducted a systematic scoping review of the empirical publications focused on cultural competence interventions for healthcare professionals in European countries. The search was conducted in eight thematic (PsycINFO, MedLine, and PubPsych) and multidisciplinary databases (Academic Search Ultimate, E-Journals, Scopus, ProQuest, and Web of Science) to identify relevant publications up to 2023. Results were presented qualitatively. Out of the initial 6506 records screened, a total of 63 publications were included. Although the interventions were implemented in 23 different European countries, cultural competence interventions have not been widely adopted in Europe. Significant heterogeneity was observed in the conception and operacionalización of cultural competence models and in the implementation of the interventions. The interventions have mostly aimed at improving healthcare for minority population groups and have focused on the racial and ethnic dimensions of the individual. Future research is needed to contribute to the conceptual development of cultural competence to design programs tailored to European superdiversity. This scoping review has been registered in OSF and is available for consultation.

Apple pomace extract: photochemopreventive action against UVA radiation in a cell culture model

Apple pomace, a byproduct from juice industry, can be used as a valuable nutraceutical, hence its phenolic compounds, including rutin, are powerful antioxidants. This work evidence the extract enriched with apple polyphenols from apple pomace using ethanol: water as solvent and partitioned with ethyl acetate (1:1 w/w), where its lyophilized product was evaluated for its photochemopreventive action on MRC-5 fibroblasts against a harmful UVA radiation dose. The enriched apple pomace extract was able to promote photochemopreventive action against UVA radiation, decreasing cell death and the lipid peroxidation formation. Therefore, the optimized extraction process may make the apple pomace application viable as a source of bioactive extracts with photochemopreventive action against ultraviolet radiation to the use in dermal formulations.

P16 Fibroblast signalling crosstalk drives junctional epidermolysis bullosa disease progression

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a devastating hereditary skin disorder characterized by severe fragility of skin and mucus membrane, emerges as blistering with little or no trauma. The major classification of JEB includes JEB intermediate and JEB severe; the latter is the most common subtype, which is the result of loss of function mutations in the skin basement membrane protein laminin-332. Lacking this protein impairs basement membrane adhesion of epidermal keratinocytes and leads to deregulated epidermal–dermal crosstalk. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their specific involvement in skin diseases is largely unknown. Here, we investigate how fibroblast subpopulations, namely papillary and reticular fibroblasts, impact JEB disease progression and skin regeneration. Methods Three-dimensional (3D) organotypic (OT) combining healthy (control) and diseased (JEB) keratinocytes cells with distinct fibroblast subpopulations from different anatomical sites were developed and analysed by immunohistochemistry and RNA-Seq. Results JEB OTs with papillary fibroblasts showed increased epidermal growth, invagination of basal layer and deregulated differentiation compared with reticular fibroblasts. RNA-Seq analysis revealed altered activity in key signalling including immune, cytokine, gap junction, mitogen-activated protein kinase and remodelling of keratinization and ECM proteins, particularly in JEB OT cultured with papillary fibroblast. JEB OTs and cells also demonstrated variation in cell proliferation and activation of transforming growth factor-β1 and immune cell signalling. Currently, we are targeting identified signalling pathways in our two-dimensional and 3D cell culture models and identifying druggable targets, before testing selected candidates in our JEB mouse disease model for further preclinical validation. Conclusions By dissecting the pathological signalling interactions between keratinocytes and distinct fibroblast subpopulations, our goal is to identify new therapeutic target to improve the skin health and regeneration in patients with JEB.

Abstract A019: TRIM29 enhances the invasion of bladder cancer cells by modulating the intermediate filament network and focal adhesions

Abstract Background: Bladder cancer patient treatment and outcomes are determined by the tumor’s ability to invade and metastasize. Our previous work has shown that TRIM29, also known as ATDC, is regulated by TP63 in basal bladder cancers, where it promotes cancer invasion and metastasis. We also observe that TRIM29 predominately localizes to intermediate filaments in invasive bladder cancer cells. Hypothesis: TRIM29 promotes the invasive progression of bladder cancer by regulating K14-containing intermediate filaments and focal adhesions. Methods: To test this hypothesis, we utilized cell culture models of bladder cancer, live cell imaging and 2D and 3D invasion assays to examine the role of TRIM29 in the regulation of K14, focal adhesions and the migration and invasion and bladder cancer cells. Results: We observed a physical interaction between TRIM29 and K14 filamentous structures in bladder cancer cells. We also found that TRIM29 and K14 regulate focal adhesion stability during bladder cancer invasion. Finally, we found that both K14 and the focal adhesion protein, zyxin (ZYX), are essential for bladder cancer migration and invasion, acting downstream of TRIM29. Conclusions: Our findings highlight a novel role for TRIM29 in regulating the cytoskeleton and focal adhesion dynamics during tumor cell invasion and suggest a potential therapeutic pathway for treating bladder cancer. Citation Format: Yin Wang, Nicole A. Jerome, Allan Kelleher, Marian L. Henderson, Mark Day, Pierre A. Coulombe, Phillip L. Palmbos. TRIM29 enhances the invasion of bladder cancer cells by modulating the intermediate filament network and focal adhesions [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A019.

Cellular resolution contributions to ictal population signals.

The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. This work provides a critical link between single neuron activity and population measures of seizure activity.

Impact of CDK Inhibitors on TBXT Expression in Chordoma Cell Lines Including the First Stable Cell Line of a High-Grade Chordoma.

Chordomas are very rare malignant neoplasms of the bone occurring almost exclusively along the spine. As the tumours are thought to arise from notochordal remnants, the vast majority of chordomas express the TBXT gene, resulting in detectable nuclear amounts of its gene product brachyury. This T-Box transcription factor is commonly recognised as being essential in chordoma cells, and limiting TBXT expression is thought to be the key factor in controlling this tumour. Although the tumour is rare, distinct molecular differences and vulnerabilities have been described with regard to its location and the progression status of the disease, rendering it mandatory for novel cell lines to reflect all relevant chordoma subtypes. Here, we describe a novel chordoma cell line arising from the pleural effusion of a disseminated, poorly differentiated chordoma. This cell line, U-CH22, represents a highly aggressive terminal chordoma and, therefore, fills a relevant gap within the panel of available cell culture models for this orphan disease. CDK7 and CDK9 inhibition was lately identified as being effective in reducing viability in four chordoma cell lines, most likely due to a reduction in brachyury levels. In this study, we determined the capability of the CDK7 inhibitor THZ1 and the CDK1/2/5/9 inhibitor dinaciclib to reduce TBXT expression at mRNA and protein levels in a broad range of nine cell lines that are models of primary, recurrent, and metastasised chordoma of the clivus and the sacrum.