Cellular Events Research Articles

Elevated reactive oxygen species can drive the alternative lengthening of telomeres pathway in ATRX-null cancers.

The alternative lengthening of telomeres (ALT) pathway is a telomerase-independent mechanism for immortalization in cancer cells and is commonly activated in low-grade and high-grade glioma, as well as osteosarcoma. The ALT pathway can be activated under various conditions and has often been shown to include mutational loss of ATRX. However, this is insufficient in isolation and so other cellular event must also be implicated. It has been shown that excessive accumulation of DNA:RNA hybrid structures (R-loops) and/or formation of DNA-protein crosslinks (DPCs) can be other important driving factors. The underlying cellular events leading to R-loop and DPC formation in ALT cancer cells to date remain unclear. Here, we demonstrate that excessive cellular reactive oxygen species (ROS) is an important causative factor in the evolution of ALT-telomere maintenance in ATRX-deficient glioma. We identified three sources of elevated ROS in ALT-positive gliomas: co-mutation of SETD2, downregulation of DRG2, and hypoxic tumour microenvironment. We demonstrate that elevated ROS leads to accumulation of R-loops and, crucially, resolution of R-loops by the enzyme RNase H1 prevents ALTpathway activity in cells exposed to elevated ROS. Further, we found a possible causal link between the formation of R-loops and the accumulation of DPCs, in particular, formation of TOP1 complexes covalently linked to DNA (Top1cc). We also demonstrate that elevation of ROS can trigger over-activity of the ALT pathway in osteosarcoma and glioma cell lines, resulting in excessive DNA damage and cell death. This work presents important mechanistic insights into the endogenous origin of excessive R-loops and DPCs in ALT-positive cancers, as well as highlighting potential novel therapeutic approaches in these difficult-to-treat cancer types.

TockyPrep: data preprocessing methods for flow cytometric fluorescent timer analysis

Background:Fluorescent Timer proteins, which display time-dependent changes in their emission spectra, are invaluable for analyzing the temporal dynamics of cellular events at the single-cell level. We previously developed the Timer-of-cell-kinetics-and-activity (Tocky) tools, utilizing a specific Timer protein, Fast-FT, to monitor temporal changes in cellular activities. Despite their potential, the analysis of Timer fluorescence in flow cytometry is frequently compromised by variability in instrument settings and the absence of standardized preprocessing methods. The development and implementation of effective data preprocessing methods remain to be achieved.Results:In this study, we introduce the R package that automates the data preprocessing of Timer fluorescence data from flow cytometry experiments for quantitative analysis at single-cell level. Our aim is to standardize Timer data analysis to enhance reproducibility and accuracy across different experimental setups. The package includes a trigonometric transformation method to elucidate the dynamics of Fluorescent Timer proteins. We have identified the normalization of immature and mature Timer fluorescence data as essential for robust analysis, clarifying how this normalization affects the analysis of Timer maturation. These preprocessing methods are all encapsulated within the TockyPrep package.Conclusions:TockyPrep is available for distribution via GitHub at https://github.com/MonoTockyLab/TockyPrep, providing tools for data preprocessing and basic visualization of Timer fluorescence data. This toolkit is expected to enhance the utility of experimental systems utilizing Fluorescent Timer proteins, including the Tocky tools.

Early spinal cord development: from neural tube formation to neurogenesis.

As one of the simplest and most evolutionarily conserved parts of the vertebrate nervous system, the spinal cord serves as a key model for understanding the principles of nervous system construction. During embryonic development, the spinal cord originates from a population ofbipotent stem cells termed neuromesodermal progenitors, which are organized within a transient embryonic structure known as the neural tube. Neural tube morphogenesis differs along its anterior-to-posterior axis: most of the neural tube (including the regions that will develop into the brain and the anterior spinal cord) forms via the bending and dorsal fusion of the neural groove, but the establishment of the posterior region of the neural tube involves de novo formation of a lumen within a solid medullary cord. The early spinal cord primordium consists of highly polarized neural progenitor cells organized into a pseudostratified epithelium. Tight regulation of the cell division modes of these progenitors drives the embryonic growth of the neural tube and initiates primary neurogenesis. A rich history of observational and functional studies across various vertebrate models has advanced our understanding of the cellular events underlying spinal cord development, and these foundational studies are beginning to inform our knowledge of human spinal cord development.

H-bonded organic frameworks as ultrasound-programmable delivery platform.

The precise control of mechanochemical activation within deep tissues using non-invasive ultrasound holds profound implications for advancing our understanding of fundamental biomedical sciences and revolutionizing disease treatments1-4. However, a theory-guided mechanoresponsive materials system with well-defined ultrasound activation has yet to be explored5,6. Here we present the concept of using porous hydrogen-bonded organic frameworks (HOFs) as toolkits for focused ultrasound (FUS) programmably triggered drug activation to control specific cellular events in the deep brain, through on-demand scission of the supramolecular interactions. A theoretical model is developed to potentially visualize the mechanochemical scission and ultrasound mechanics, providing valuable guidelines for the rational design of mechanoresponsive materials to achieve programmable control. To demonstrate the practicality of this approach, we encapsulate the designer drug clozapine N-oxide (CNO) into the optimal HOF nanocrystals for FUS-gated release to activate engineered G-protein-coupled receptors in the ventral tegmental area (VTA) of mice and rats and hence achieve targeted neural circuit modulation even at depth 9 mm with a latency of seconds. This work demonstrates the capability of ultrasound to precisely control molecular interactions and develops ultrasound-programmable HOFs to non-invasively and spatiotemporally control cellular events, thereby facilitating the establishment of precise molecular therapeutic possibilities.

Environmental conditions controlling the morphology of shallow orographic convection

Abstract Quasi-stationary rainbands capable of producing heavy localized precipitation have been observed over the Oregon Coastal Range and other mesoscale mountain ridges. These bands thus present an important forecasting problem, which is challenged by the inability of operational NWP models to accurately resolve them. To aid prediction of these events, this study synthesizes an observational climatology with idealized large-eddy simulations of shallow convection over the Coastal Range. The climatology identified cases with banded and cellular morphologies over the Coastal Range and determined composite upstream soundings for each morphology. While prominent differences in these soundings included stronger low-level winds and dry static stability in the banded events, these differences alone did not fully determine the resulting cloud morphology in the simulations. Another key factor was the turbulence intensity in the impinging atmospheric boundary layer (ABL), which is partially controlled by the sea–air temperature difference over the eastern Pacific Ocean (ΔTSA). While banded events mostly exhibit ΔTSA < 0 and weak ABL turbulence, cellular events mostly exhibit the opposite. ABL turbulence was thus hypothesized to favor cells by disrupting the lee-wave circulations responsible for organizing the bands. A new parameter R was developed to predict cloud morphology based on the ratio of the TKE of transient turbulent velocity fluctuations to that of stationary lee-wave perturbations. This parameter accurately predicted the cloud morphology in numerous simulations with varying upstream flows and ΔTSA. It also provides a simple explanation for why the observed characteristics of banded events (stronger low-level winds and ABL stabilities, ΔTSA < 0) all favor band development.

A mechanism-informed deep neural network enables prioritization of regulators that drive cell state transitions

Cells are regulated at multiple levels, from regulations of individual genes to interactions across multiple genes. Some recent neural network models can connect molecular changes to cellular phenotypes, but their design lacks modeling of regulatory mechanisms, limiting the decoding of regulations behind key cellular events, such as cell state transitions. Here, we present regX, a deep neural network incorporating both gene-level regulation and gene-gene interaction mechanisms, which enables prioritizing potential driver regulators of cell state transitions and providing mechanistic interpretations. Applied to single-cell multi-omics data on type 2 diabetes and hair follicle development, regX reliably prioritizes key transcription factors and candidate cis-regulatory elements that drive cell state transitions. Some regulators reveal potential new therapeutic targets, drug repurposing possibilities, and putative causal single nucleotide polymorphisms. This method to analyze single-cell multi-omics data demonstrates how the interpretable design of neural networks can better decode biological systems.

Al-Based Metal-Organic Nanoslice-Coupled Phospholipid Layer Enables Highly Enriched Biological External Vesicles.

Systematic analysis of the information content of biological external vesicles (BEVs) is essential for understanding the complex relationships between metabolic processes and cells at a systemic level. Although considerable efforts have been made to enrich BEVs, their comprehensive analysis in a way that maintains and stabilizes information maintenance at high doses remains a challenge. To address this issue, we developed a metal-organic nanoslice-coupled exocytosis phospholipid layer method that utilized subtle interactions between the metal-organic nanoslice and the phospholipid molecular layer of the membrane to achieve the convenient and efficient enrichment of BEVs. The method bypassed the conventional ultracentrifugation and size exclusion separation and helped to highly preserve the internal information on BEVs. By integrating and analyzing the enriched BEVs for elements such as membrane proteins and endotoxins, the simplicity and robustness of the metal-organic nanoslice-coupled vesicle surface phospholipid layer technology were verified, which provided a reliable platform for studying the cellular events associated with outer vesicles and expanded the biological applications of the metal-organic nanoslice.

Polyamines enhance repeat-associated non-AUG translation from CCUG repeats by stabilizing the tertiary structure of RNA.

Repeat expansion disorders are caused by abnormal expansion of microsatellite repeats. Repeat-associated non-AUG (RAN) translation is one of the pathogenic mechanisms underlying repeat expansion disorders, but the exact molecular mechanism underlying RAN translation remains unclear. Polyamines are ubiquitous biogenic amines that are essential for cell proliferation and cellular functions. They are predominantly found in cells in complexes with RNA and influence many cellular events, but the relationship between polyamines and RAN translation is yet to be explored. Here, we show that, in both a cell-free protein synthesis system and cell culture, polyamines promote RAN translation of RNA containing CCUG repeats. The CCUG-dependent RAN translation is suppressed when cells are depleted of polyamines but can be recovered by the addition of polyamines. Thermal stability analysis revealed that the tertiary structure of the CCUG-repeat RNA is stabilized by the polyamines. Spermine was the most effective polyamine for stabilizing CCUG-repeat RNA and enhancing RAN translation. These results suggest that polyamines, particularly spermine, modulate RAN translation of CCUG-repeat RNA by stabilizing the tertiary structure of the repeat RNA.

Remote Ischemic Conditioning Attenuates Transneuronal Degeneration and Promotes Stroke Recovery via CD36-Mediated Efferocytosis.

Remote ischemic conditioning (RIC) has been implicated in cross-organ protection in cerebrovascular disease, including stroke. However, the lack of a consensus protocol and controversy over the clinical therapeutic outcomes of RIC suggest an inadequate mechanistic understanding of RIC. The current study identifies RIC-induced molecular and cellular events in the blood, which enhance long-term functional recovery in experimental cerebral ischemia. Naive mice or mice subjected to transient ischemic stroke were randomly selected to receive sham conditioning or RIC in the hindlimb at 2 hours post-stroke. At 3 days post-stroke, monocyte composition in the blood was analyzed, and brain tissue was examined for monocyte-derived macrophage (Mφ), levels of efferocytosis, and CD36 expression. Mouse with a specific deletion of CD36 in monocytes/Mφs was used to establish the role of CD36 in RIC-mediated modulation of efferocytosis, transneuronal degeneration, and recovery following stroke. RIC applied 2 hours after stroke increased the entry of monocytes into the injured brain. In the postischemic brain, Mφ had increased levels of CD36 expression and efferocytosis. These changes in brain Mφ were derived from RIC-induced changes in circulating monocytes. In the blood, RIC increased CD36 expression in circulating monocytes and shifted monocytes to a proinflammatory LY6CHigh state. Conditional deletion of CD36 in Mφ abrogated the RIC-induced monocyte shift in the blood and efferocytosis in the brain. During the recovery phase of stroke, RIC rescued the loss of the volume and of tyrosine hydroxylase+ neurons in substantia nigra and behavioral deficits in wild-type mice but not in mice with a specific deletion of CD36 in monocytes/Mφs. RIC induces a shift in monocytes to a proinflammatory state with elevated CD36 levels, and this is associated with CD36-dependent efferocytosis in Mφs that rescues delayed transneuronal degeneration in the postischemic brain and promotes stroke recovery. Together, these findings provide novel insight into our mechanistic understanding of how RIC improves poststroke recovery.

Cellular crosstalk promotes hepatic progenitor cell proliferation and stellate cell activation in 3D co-culture.

Following liver damage, ductular reaction often coincides with liver fibrosis. Proliferation of hepatic progenitor cells is observed in ductular reaction, while activated hepatic stellate cells (HSCs) are the main drivers of liver fibrosis. These observations may suggest a functional interaction between these two cell types. Here we report on an in vitro co-culture system to examine these interactions and validate their co-expression in human liver explants. In a 3D organoid co-culture system we combined freshly isolated quiescent mouse HSCs and fluorescently labeled progenitor cells (undifferentiated intrahepatic cholangiocyte organoids), permitting real-time observation of cell morphology and behavior. After 7 days, cells were sorted based on the fluorescent label and analyzed for changes in gene expression. In the 3D co-culture system, the proliferation of progenitor cells is enhanced and HSCs are activated, recapitulating the cellular events observed in the patient liver. Both effects in 3D co-culture require close contact between the two different cell types. HSC activation during 3D co-culture differs from quiescent (3D mono-cultured) HSCs and activated HSCs on plastic (2D mono-culture). Upregulation of a cluster of genes containing Aldh1a2, Cthrc1 and several genes related to frizzled binding/ Wnt signaling were exclusively observed in 3D co-cultured HSCs. The localized co-expression of specific genes was confirmed by spatial transcriptomics in human liver explants. An in vitro 3D co-culture system provides evidence for direct interactions between HSCs and progenitor cells, which are sufficient to drive responses that are similar to those seen during ductular reaction and fibrosis. This model paves the way for further research into the cellular basis of liver pathology.

Suppressing APOE4-induced neural pathologies by targeting the VHL–HIF axis

The ε4 variant of human apolipoprotein E (APOE4) is a key genetic risk factor for neurodegeneration in Alzheimer's disease and elevated all-cause mortality in humans. Understanding the factors and mechanisms that can mitigate the harmful effects of APOE4 has significant implications. In this study, we find that inactivating the VHL-1 (Von Hippel-Lindau) protein can suppress mortality, neural and behavioral pathologies caused by transgenic human APOE4 in Caenorhabditis elegans. The protective effects of VHL-1 deletion are recapitulated by stabilized HIF-1 (hypoxia-inducible factor), a transcription factor degraded by VHL-1. HIF-1 activates a genetic program that safeguards against mitochondrial dysfunction, oxidative stress, proteostasis imbalance, and endolysosomal rupture-critical cellular events linked to neural pathologies and mortality. Furthermore, genetic inhibition of Vhl reduces cerebral vascular injury and synaptic lesions in APOE4 mice, suggesting an evolutionarily conserved mechanism. Thus, we identify the VHL-HIF axis as a potent modulator of APOE4-induced neural pathologies and propose that targeting this pathway in nonproliferative tissues may curb cellular damage, protect against neurodegeneration, and reduce tissue injuries and mortality.

Tepary Bean (Phaseolus acutifolius) Lectins as Modulators of Intracellular Calcium Mobilization in Breast Cancer and Normal Breast Cells

Lectins are proteins that specifically recognize carbohydrates on cell membranes, triggering several cellular events such as apoptosis of cancer-transformed cells; however, the mechanisms of action remain incompletely understood. Our research group has reported that a concentrated fraction of Tepary bean lectins (Phaseolus acutifolius; TBLF) exhibits the concentration-dependent induction of apoptosis in colon cancer cells by caspase activation. It is well established that an increase in cytoplasmic calcium ([Ca2+]i) initiates intracellular signals involved in processes such as exocytosis, gene transcription, apoptosis, cell cycle regulation, and muscle contraction, among others. Furthermore, dysregulated calcium signaling has been implicated in various diseases, including certain neurological disorders and cancer. In this study, we aim to demonstrate the effects of native TBLF lectins and a recombinant lectin (rTBL-1) on calcium mobility in breast cancer cells (MCF-7) and non-cancerous cells (MCF-12F). Both TBLF and rTBL-1 increased intracellular calcium concentrations and mobilized calcium from intracellular stores in a concentration-dependent manner; however, the two cell lines exhibited differential responses. While MCF-12F cells restored cytoplasmic calcium concentration, MCF-7 cells maintained a high intracellular calcium concentration. This strongly suggests that lectins can elicit differential cellular responses in cancer and non-cancer cells due to variations in their intrinsic mechanisms of calcium homeostasis. Finally, we demonstrated that TBLF and rTBL-1 can differentially alter Metabolic Cellular Activity (MCA) as a direct measure of cell viability (CVi) in both cell lines. These findings strengthen the evidence of the therapeutic potential of Tepary bean lectins. Undoubtedly, further studies will be necessary to elucidate the mechanisms underlying their applications.

TRAF1 promotes osteoclastogenesis by enhancing metabolic adaptation to oxidative phosphorylation in an AKT-dependent manner.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a crucial signaling adaptor involved in multiple cellular events. However, its role in regulating osteoclastogenesis and energy metabolism remains unclear. Here, we report that TRAF1 promotes osteoclastogenesis and oxidative phosphorylation (OXPHOS). Employing RNA-sequencing, we found that TRAF1 is markedly upregulated during osteoclastogenesis and is positively associated with osteoporosis. TRAF1 knockout inhibits osteoclastogenesis and increases bone mass in both normal and ovariectomized adult mice, without affecting bone mass in childhood. Furthermore, TRAF1 promotes osteoclast OXPHOS by increasing the phosphorylation level of AKT. Mechanistically, TRAF1 functions to inhibit TRAF2-induced ubiquitination of Gβl, a known activator of AKT, and further upregulates AKT phosphorylation. Rescue experiments revealed that the inhibitory effects of TRAF1 knockout on osteoclastogenesis, OXPHOS, and bone mass are dependent on AKT. Collectively, our findings uncover a previously unrecognized function of TRAF1 in regulating osteoclastogenesis and energy metabolism, and establish a novel TRAF1-AKT-OXPHOS axis in osteoclasts.

Tremor in the Age of Omics: An Overview of the Transcriptomic Landscape of Essential Tremor.

Essential Tremor (ET) is the most common movement disorder and has a worldwide prevalence of 1%, including 5% of the population over 65 years old. It is characterized by an active, postural or kinetic tremor, primarily affecting the upper limbs, and is diagnosed based on clinical characteristics. The pathological mechanisms of ET, however, are mostly unknown. Moreover, despite its high heritability, genetic studies of ET genetics have yielded mixed results. Transcriptomics is a field that has the potential to reveal valuable insights about the processes and pathogenesis of ET thus providing an avenue for the development of more effective therapies. With the emergence of techniques such as single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq), molecular and cellular events can now be more closely examined, providing valuable insights into potential causal mechanisms. In this review, we review the growing literature on transcriptomic studies in ET, aiming to identify biological pathways involved and explore possible avenues for further ET research. We emphasized the convergence on shared of biological pathways across several studies, specifically axonal guidance and calcium signaling. These findings posit multiple hypotheses linking both pathways through the regulation of axonal and synaptic plasticity. We conclude that increasing the sample size is vital to uncover the subtleties of ET clinical and pathological heterogeneity. Additionally, integrating Multiomics approaches should provide a comprehensive understanding of the disease's pathophysiology.

Molecular Insights into Fungal Innate Immunity Using the Neurospora crassa - Pseudomonas syringae Model.

Recent comparative genomics and mechanistic analyses support the existence of a fungal immune system. Fungi encode genes with features similar to non-self recognition systems in plants, animals, and bacteria. However, limited functional or mechanistic evidence exists for the surveillance-system recognition of heterologous microbes in fungi. We found that Neurospora species coexist with Pseudomonas in their natural environment. We leveraged two model organisms, Neurospora crassa and Pseudomonas syringae DC3000 (PSTDC3000) to observe immediate fungal responses to bacteria. PSTDC3000 preferentially surrounds N. crassa cells on a solid surface, causing environmental dependent growth responses, bacterial proliferation and varying fungal fitness. Specifically, the Type III secretion system (T3SS) ΔhrcC mutant of PSTDC3000 colonized N. crassa hyphae less well. To dissect initial cellular signaling events within the population of germinated asexual spores (germlings), we performed transcriptomics on N. crassa after PSTDC3000 inoculation. Upon contact with live bacteria, a subpopulation of fungal germlings initiate a response as early as ten minutes post-contact revealing transcriptional differentiation of Reactive Oxygen Species (ROS) mechanisms, trace metal warfare, cell wall remodeling dynamics, multidrug-efflux transporters, secondary metabolite synthesis, and excretion. We dissected mutants of plausible receptors, signaling pathways, and responses that N. crassa uses to detect and mount a defense against PSTDC3000 and found seven genes that influence resistant and susceptibility phenotypes of N. crassa to bacterial colonization. Mutants in genes encoding a ctr copper transporter ( tcu-1 ), ferric reductase ( fer-1 ), superoxide reductase ( sod-2 ), multidrug resistance transporter ( mdr-6 ), a secreted lysozyme-Glycoside hydrolase ( lyz ) and the Woronin body tether leashin (NCU02793, lah-1 and lah-2 ) showed a significant reduction of growth in the presence of bacteria, allowing the bacteria to fully take over the fungal mycelium faster than wildtype. In this study we provide a bacterial-fungal model system within Dikarya that allows us to begin to dissect signaling pathways of the putative fungal immune system.

MLL4 regulates postnatal palate growth and midpalatal suture development

MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the MLL4 gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the Mll4-cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in Mll4-cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in Mll4-cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the Mll4-cKO mice emerged as early as 1 day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as Col2a1 and Acan, at birth. Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the Mll4-cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in Mll4-cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.

In situ vaccine "seeds" for enhancing cancer immunotherapy by exploiting apoptosis-associated morphological changes.

Despite the development of many effective immunoadjuvants (IAs), the therapeutic efficacy of in situ vaccines for anti-tumor applications remains limited. Inspired by the morphological changes occurring during apoptosis, this study aims to leverage the release process of autologous tumor antigens (ATAs) to enhance the anti-tumor activity of in situ vaccines. We developed five distinct liposomes, each with unique characteristics and functions, incorporating FDA-approved monophosphoryl lipid A (MPLA) adjuvants into their lipid bilayers. Our findings revealed that the apoptotic bodies generated from tumor cells treated with membrane-fusion liposomes (MFLs) exhibited a greater capacity for immune activation. Mechanistic studies demonstrated that MFLs can utilize the morphological changes associated with apoptosis to accurately deliver adjuvants to apoptotic bodies. To further optimize the efficiency of antigen presentation by these apoptotic bodies as an adjuvant redistribution platform, we encapsulated a calcium chelator within the MFLs to inhibit the externalization of phosphatidylserine (PS) during apoptosis. Through a series of apoptosis-related cellular events, the vaccine can widely disseminate immunoadjuvants (IAs) within tumor tissues, similar to the dispersal of plant seeds. To the best of our knowledge, this is the first approach to utilize apoptosis-associated morphological changes to enhance the immunotherapeutic efficacy of cancer vaccines.

MicroRNAs in Parkinson's disease: From pathogenesis to diagnostics and therapeutic strategies.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by pathological changes, including the loss of dopaminergic neurons and abnormal aggregation of α-synuclein (α-syn). Certain cellular and molecular events are involved; however, the origin and significance of these events remain uncertain. The discovery of microRNAs (miRNAs) predicted to play a pivotal role in various regulatory processes has emerged. Studies on the dysregulation of miRNAs in PD pathogenesis, diagnosis, and treatment have recently gained attention. This review aims to encapsulate recent research developments concerning the function of miRNAs in the pathophysiology of PD and their prospective applications as diagnostic and therapeutic biomarkers, targets, and pharmaceuticals. The most effective drug delivery approach for the treatment of PD, transnasal-cerebral drug delivery, has also been briefly described. The advantage of this delivery strategy is its capacity to bypass the blood-brain barrier, enabling direct administration of medication to the brain, which improves therapeutic efficacy and minimizes side effects.

OP24 TL1A-activated T cells as upstream regulators of perianal fistulizing disease-associated changes in the rectum of Crohn's Disease patients

Abstract Background About 25% of Crohn’s disease (CD) patients develop perianal fistulizing disease (PFD), the pathogenesis of which remains poorly understood. Here we aimed to characterise the molecular and cellular events underlying this complication. Methods Non-inflamed rectal biopsies from CD patients with or without PFD (CD-PFD and CD) were processed for single-cell (n=14) or bulk (n=20) RNA analysis. Functional experiments were conducted on human primary intestinal fibroblasts and peripheral T cells. Results Single-cell RNA sequencing revealed the increased abundance of some cell types, such as intestinal fibroblasts, in the CD-PFD rectal mucosa relative to CD (Fig. 1A). Within the stromal subset, the lamina propria S1 fibroblasts showed the highest number of differentially expressed genes (DEGs) between CD and CD-PFD patients, including an upregulation of TFPI2 and a downregulation of the TGFβ-responding gene CTGF (Fig.1B). These changes were validated by bulk RNA analysis, where we also observed overexpression of the previously described PFD target genes MMP1 and MMP3 (Fig.1C). In vitro studies with intestinal fibroblasts confirmed the negative regulation by TGFβ of the PFD-signature, thus ruling out TGFβ as a driver of the PFD-associated fibroblast signature (Fig.1D). Next, we investigated DEGs in the lymphoid compartment and identified lymphotoxin beta (LTB) as being upregulated in CD-PFD intestinal CD4+ T cells (Fig.1E). LTB interacts with its receptor LTBR, which is expressed in stromal, epithelial and myeloid cells. Interestingly, LTB-LTBR interactions were increased in the CD-PFD group. In vitro studies in primary fibroblasts showed that LTB upregulates TFPI2, while it downregulates CTGF, thus reproducing the PFD-associated signature (Fig.1F). Finally, we submitted the top 50 genes overexpressed by CD-PFD CD4+ T cells to the immune dictionary1, which identified TL1A as its most likely driver (Fig.2A). In line with this, the TL1A receptor TNFRSF25 was overexpressed in CD-PFD CD4+ T cells (Fig.2B). As a link between TL1A and LTB is not described in the literature, we stimulated anti-CD3-activated peripheral T cells with TL1A and observed the significant upregulation of LTa1b2, the functional form of LTB (Fig.2C). Intriguingly, we also observed that while TNF can drive the PFD-associated signature in fibroblasts, it cannot induce LTa1b2 in T cells (Fig.2D), suggesting that TNF and TL1A/LTB signalling are two independent but potentially complementary pathways in PFD. Conclusion In summary, we propose a previously unrecognized TL1A-LTB axis as being responsible for inducing PFD-associated alterations in the rectal mucosa (Fig.2E), and we hypothesise that inhibition of TL1A might be worth exploring in the context of perianal disease.

Anti-cancer effect of midazolam via downregulating YWHAH in papillary thyroid cancer cells

The work is aimed to investigate whether midazolam functions in thyroid cancer and reveal the potential mechanism of action. Cell viability was detected by CCK-8 method when treated by varying doses of midazolam to detect the cytotoxicity of midazolam on human thyroid follicular epithelial cell line and thyroid cancer cell lines. In thyroid cancer cells, EDU staining, wound healing and transwell assays were respectively used to detect cell proliferation, migration and invasion. Western blot was used to detect the expressions of matrix metalloproteinases (MMPs). Flow cytometry assay, western blot and immunofluorescence staining were used to detect cell apoptosis. CB-Dock2 server predicted midazolam-tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) interaction and western blot was also used to detect YWHAH expression. Midazolam dose-dependently decreased the viability of thyroid cancer cells and demonstrated no cytotoxicity on Nthy-ori-3-1 cells. In addition, increasing concentrations of midazolam or silencing of YWHAH significantly inhibited thyroid cancer cell proliferation, migration and invasion and induced cell apoptosis. Midazolam had a molecular binding with YWHAH and midazolam downregulated YWHAH expression. YWHAH partially reversed the impacts of midazolam on the cellular events in thyroid cancer. Collectively, midazolam may act as an anti-thyroid cancer agent via its interrelation with YWHAH.