Cellular Functions Research Articles

Quantifying Gq Signaling Using the IP1 Homogenous Time-Resolved Fluorescence (HTRF) Assay.

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and can regulate several cellular functions such as proliferation, secretion, protein expression, and cellular metabolism. Coupling of GPCRs to members of the Gq/11 protein family results in activation of inositol trisphosphate (IP3) and accumulation of calcium intracellularly. This protocol chapter outlines a step-by-step guide for utilizing the inositol phosphate-1 (IP1) accumulation assay, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, to investigate Gq-IP3 signaling. The assay serves as a valuable tool for those conducting pharmacological investigations and compound screening targeting this critical cellular pathway. This protocol chapter covers experimental setup, sample preparation, and data analysis, providing researchers with an in-depth guide to explore the pharmacology of Gq-coupled receptors.

Measuring Calcium Signaling at the Primary Cilia.

Cellular signaling is nature's ingenious way for cells to perceive their surroundings and transmit external cues to internal compartments. Due to its critical role in cellular functions, the intricate machinery of molecular signaling has been intensively studied. A diverse arsenal of techniques exists to quantify the molecules involved in these processes. Among them, calcium stands out as a ubiquitous signaling molecule with roles in countless biological pathways. To elucidate its function as a second messenger, methods for measuring intracellular calcium have steadily evolved. This chapter introduces various methods for investigating calcium signaling cascades in cells as well as in cilia (thin hairlike projections) specifically, where calcium signaling is triggered by different cilial manipulation techniques.

Active RNA synthesis patterns nuclear condensates.

Biomolecular condensates are membraneless compartments that organize biochemical processes in cells. In contrast to well-understood mechanisms describing how condensates form and dissolve, the principles underlying condensate patterning - including their size, number and spacing in the cell - remain largely unknown. We hypothesized that RNA, a key regulator of condensate formation and dissolution, influences condensate patterning. Using nucleolar fibrillar centers (FCs) as a model condensate, we found that inhibiting ribosomal RNA synthesis significantly alters the patterning of FCs. Physical theory and experimental observations support a model whereby active RNA synthesis generates a non-equilibrium state that arrests condensate coarsening and thus contributes to condensate patterning. Altering FC condensate patterning by expression of the FC component TCOF1 impairs ribosomal RNA processing, linking condensate patterning to biological function. These results reveal how non-equilibrium states driven by active chemical processes regulate condensate patterning, which is important for cellular biochemistry and function.

Endonuclease A in Streptococcus pneumoniae: escaping from neutrophil extracellular traps (NETs) and relationship in immunogenicity

Streptococcus pneumoniae (S. pneumoniae), which is a Gram-positive diplococcus, has emerged as a significant human pathogen. It is a primary cause of bacterial pneumonia, otitis media, meningitis, and septicemia, leading to a considerable impact on global morbidity and mortality. The investigation of S. pneumoniae and its virulence factors has resulted in the identification of surface endonuclease A (EndA). EndA functions in DNA uptake during natural transformation and plays a significant role in gene transfer. The ability of S. pneumoniae to degrade neutrophil extracellular traps (NETs) enhances its virulence and invasive potential in pneumococcal infections. NETosis occurs when neutrophils release chromatin into the extracellular space to form NETs, capturing and neutralizing pathogens. Currently, NETosis can be induced by several microbes, particulate matter, and sterile stimuli through distinct cellular mechanisms, and this includes the involvement of EndA in S. pneumoniae. Here, we reviewed the cellular functions of EndA, its role in S. pneumoniae as a virulence factor in relation to NETosis, its relationship to immunogenicity, and its involvement in several diseases. The discovery of this relationship would significantly impact therapeutic technology in reducing disease burden, especially pneumococcal infections.

A microphysiological system for studying barrier health of live tissues in real time

Epithelial cells create barriers that protect many different components in the body from their external environment. Increased gut barrier permeability (leaky gut) has been linked to several chronic inflammatory diseases. Understanding the cause of leaky gut and effective interventions are elusive due to the lack of tools that maintain tissue’s physiological environment while elucidating cellular functions under various stimuli ex vivo. Here we present a microphysiological system that records real-time barrier permeability of mouse colon in a physiological environment over extended durations. The system includes a microfluidic chamber; media composition that preserves microbiome and creates necessary oxygen gradients across the barrier; and integrated sensor electrodes for acquiring transepithelial electrical resistance (TEER). Our results demonstrate that the system can maintain tissue viability for up to 72 h. The TEER sensors can distinguish levels of barrier permeability when treated with collagenase and low pH media and detect different thickness in the tissue explant.

Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC.

The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca2+-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.

Length-sensitive partitioning of Caenorhabditis elegans meiotic chromosomes responds to proximity and number of crossover sites

Sensing and control of size are critical for cellular function and survival. A striking example of size sensing occurs during meiosis in the nematode Caenorhabditis elegans. C.elegans chromosomes compare the lengths of the two chromosome "arms" demarcated by the position of their single off-center crossover, and they differentially modify these arms to ensure that sister chromatid cohesion is lost specifically on the shorter arm in the first meiotic division, while the longer arm maintains cohesion until the second division. While many of the downstream steps leading to cohesion loss have been characterized, the length-sensing process itself remains poorly understood. Here, we have used cytological visualization of short and long chromosome arms, combined with quantitative microscopy, live imaging, and simulations, to investigate the principles underlying length-sensitive chromosome partitioning. By quantitatively analyzing short-arm designation patterns on fusion chromosomes carrying multiple crossovers, we develop a model in which a short-arm-determining factor originates at crossover designation sites, diffuses within the synaptonemal complex, and accumulates within crossover-bounded chromosome segments. We demonstrate experimental support for a critical assumption of this model: that crossovers act as boundaries to diffusion within the synaptonemal complex. Further, we develop a discrete simulation based on our results that recapitulates a wide variety of observed partitioning outcomes in both wild-type and previously reported mutants. Our results suggest that the concentration of a diffusible factor is used as a proxy for chromosome length, enabling the correct designation of short and long arms and proper segregation of chromosomes.

Biogenesis of omegasomes and autophagosomes in mammalian autophagy.

Autophagy is a highly conserved catabolic pathway that maintains cellular homeostasis by promoting the degradation of damaged or superfluous cytoplasmic material. A hallmark of autophagy is the generation of membrane cisternae that sequester autophagic cargo. Expansion of these structures allows cargo to be engulfed in a highly selective and exclusive manner. Cytotoxic stress or starvation induces the formation of autophagosomes that sequester bulk cytoplasm instead of selected cargo. This rather nonselective pathway is essential for maintaining vital cellular functions during adverse conditions and is thus a major stress response pathway. Both selective and nonselective autophagy rely on the same molecular machinery. However, due to the different nature of cargo to be sequestered, the involved molecular mechanisms are fundamentally different. Although intense research over the past decades has advanced our understanding of autophagy, fundamental questions remain to be addressed. This review will focus on molecular principles and open questions regarding the formation of omegasomes and phagophores in nonselective mammalian autophagy.

Adapting to change: resolving the dynamic and dual roles of NCK1 and NCK2.

Adaptor proteins play central roles in the assembly of molecular complexes and co-ordinated activation of specific pathways. Through their modular domain structure, the NCK family of adaptor proteins (NCK1 and NCK2) link protein targets via their single SRC Homology (SH) 2 and three SH3 domains. Classically, their SH2 domain binds to phosphotyrosine motif-containing receptors (e.g. receptor tyrosine kinases), while their SH3 domains bind polyproline motif-containing cytoplasmic effectors. Due to these functions being established for both NCK1 and NCK2, their roles were inaccurately assumed to be redundant. However, in contrast with this previously held view, NCK1 and NCK2 now have a growing list of paralog-specific functions, which underscores the need to further explore their differences. Here we review current evidence detailing how these two paralogs are unique, including differences in their gene/protein regulation, binding partners and overall contributions to cellular functions. To help explain these contrasting characteristics, we then discuss SH2/SH3 structural features, disordered interdomain linker regions and post-translational modifications. Together, this review seeks to highlight the importance of distinguishing NCK1 and NCK2 in research and to pave the way for investigations into the origins of their interaction specificity.

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C)

Abstract Background Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare and severe complication of SARS-CoV-2 infection that is characterized by multi-organ involvement and substantial inflammation. The immunological responses in MIS-C are not fully known. Antibody-mediated viral clearance in which innate immune cells interact with antibodies via their Fc receptors to remove the virus is an important mechanism for reducing SARS-CoV-2 viral load. However, little is known about how antibody-mediated cellular responses in MIS-C compared to acute COVID-19 infection and direct testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Therefore, we focused on the antibody-mediated cellular responses in MIS-C relative to children and adults that had acute COVID-19 infection with spectrum of symptoms. We hypothesized that cells that bind antibodies to help clear the virus are dysfunctional in MIS-C patients, preventing the virus from being fully cleared. Methods We collected samples from MIS-C children along with pediatric and adult subjects with a clinical spectrum of COVID-19 symptoms and uninfected controls. Through these controls, we can compare Fc-mediated antibody functions between ages and clinical symptoms. We utilized high dimension flow cytometry to assess phenotype and function of innate immune cells that bind to antibodies and sequencing to study Fc receptor genetic. This data was then correlated with matched clinical data. Results Monocytes in MIS-C were hyperfunctional for antibody-dependent cellular phagocytosis and production of proinflammatory cytokines. In contrast, natural killer (NK) cells in MIS-C were hypofunctional for antibody-dependent cellular cytotoxicity and cytokine production. To provide some preliminary insight into mechanism, we show multiple ways leading to decreased cytotoxicity for NK cells, including phenotypic exhaustion of NK cells, variants in CD16 (NK cell Fc receptor) genetics that reduce Fc binding to IgG, and cytokine-induced associations. Using a novel anti-CD16 novel bispecific reagent that we developed, we could rescue the hypofunctional antibody-mediated NK cell function in MIS-C to the same levels as control. Conclusion Together, our results reveal dysregulation in antibody-mediated responses that are unique to MIS-C and may contribute to the immune pathology of this disease. Specifically, we find that monocytes are hyperfunctional with NK cells are hypofunctional. In the case of the MIS-C antibody-mediated response, while it is likely that multiple pathogenetic mechanisms contribute to MIS-C, our data suggest an imbalance of the antibody-mediated cellular response as a mechanistic correlate of this rare, but life-threatening outcome of SARS-COV-2 in children. Our results highlight that targeted therapies to reduce viral burden and/or boost NK cells responses while decreasing cytokine levels may be effective in preventing MIS-C.

NK Cell and Monocyte Dysfunction in Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multiorgan involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong Ab production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 wk postinfection. Therefore, we hypothesized that dysfunctional cell-mediated Ab responses downstream of Ab production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, whereas NK cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function invitro. Taken together, our results reveal dysregulation in Ab-mediated cellular responses of myeloid and NK cells that likely contribute to the immune pathology of this disease.

Unveiling the pan-cancer landscape of S100A16: A comprehensive analysis of prognostic significance, drug sensitivity, and immunomodulatory roles.

Accumulating evidence supports the notion that S100A16 exhibits differential expression in many human cancers, affecting cellular functions associated with tumorigenesis through various signaling pathways. While extensive research has been conducted on S100A16 in specific cancer types, a comprehensive evaluation of its role across diverse cancers remains lacking. To explore the prognostic significance, drug sensitivity, and immunomodulatory roles of S100A16, a thorough analysis was conducted at a pan-cancer level using multiple databases. Our findings revealed high expression of S100A16 RNA in various human cancers. Importantly, this elevated expression was linked to disease prognosis and drug sensitivity across a spectrum of cancers. Genetic alterations in S100A16 were characterized across multiple cancer types, and a confirmed correlation was observed in the prognosis of skin cutaneous melanoma (SKCM). Furthermore, our study demonstrated a significant association between S100A16 expression and the infiltrating levels of diverse cell types in the tumor microenvironment (TME), suggesting its potential as a prognosis predictor for immunotherapy. Novel collections of miRNAs, such as has-miR-423-5p, has-miR-769-5p, has-miR-151a-3p, and has-miR-550a-5p, targeting S100A16 at a pan-cancer level were predicted through various databases. These findings contribute to a comprehensive understanding the role of S100A16 in prognosis prediction, chemotherapy, and immunotherapy, providing valuable insights for identifying novel targets in cancer treatment.

Crowder Chain Length Variability and Excluded Volume Effect on the Phase Separation Behavior of Mucin.

Phase separation within cellular membranes, a critical process underpinning diverse cellular functions, is significantly influenced by transmembrane proteins. Therefore, elucidating the behavior of a transmembrane protein in its phase-separated state is of utmost importance. Our study explores mucin behavior in the cellular milieu, aiming to determine the role of crowder chain length and excluded volume in phase separation. Confocal microscopy images demonstrate the strong partitioning of mucin into the condensed phase influenced by hydrophobic and electrostatic interactions. Fluorescence recovery after photobleaching analysis revealed increased mobility in the presence of shorter chain length crowders, indicating the dynamic behavior of protein within condensed phases. Excluded volume calculation using the theoretical model emphasizes its importance in mucin phase separation under crowded conditions. Our findings underscore the ability of mucin to phase-separate under crowded conditions, highlighting the crucial role of excluded volume and enhancing our understanding of its involvement in cancer progression.

The DNA Damage Repair Function of Fission Yeast CK1 Involves Targeting Arp8, a Subunit of the INO80 Chromatin Remodeling Complex.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells increased double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and nonhomologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. To understand how Hhp1 and Hhp2 promote DNA damage repair, we identified new substrates of these enzymes using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 important for DNA repair. Our data suggest that Hhp1 and Hhp2 facilitate DNA repair by phosphorylating multiple substrates, including Arp8.

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy

BackgroundStress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson’s disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear.MethodsMitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.ResultsAcute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.ConclusionsPathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.

Predicting protein complexes in protein interaction networks using Mapper and graph convolution networks

Protein complexes are groups of interacting proteins that are central to multiple biological processes. Studying protein complexes can enhance our understanding of cellular functions and malfunctions and thus support the development of effective disease treatments. High-throughput experimental techniques allow the generation of large-scale protein-protein interaction datasets. Accordingly, various computational approaches to predict protein complexes from protein-protein interactions were presented in the literature. They are typically based on networks in which nodes and edges represent proteins and their interactions, respectively. State-of-the-art approaches mainly rely on clustering static networks to identify complexes. However, since protein interactions are highly dynamic in nature, recent approaches seek to model such dynamics by typically integrating gene expression data and identifying protein complexes accordingly. We propose MComplex, a method that utilizes time-series gene expression with interaction data to generate a temporal network which is passed to a generative adversarial network whose generator is a graph convolutional network. This creates embeddings which are then analyzed using a modified graph-based version of the Mapper algorithm to predict corresponding protein complexes. We test our approach on multiple benchmark datasets and compare identified complexes against gold-standard protein complex datasets. Our results show that MComplex outperforms existing methods in several evaluation aspects, namely recall and maximum matching ratio as well as a composite score covering aggregated evaluation measures. The code and data are available for free download from https://github.com/LeonardoDaou/MComplex.

A simulation study on the role of mitochondria-sarcoplasmic reticulum Ca2+ interaction in cardiomyocyte energetics during exercise.

Previous studies demonstrated that the mitochondrial Ca2+ uniporter MCU and the Na+-Ca2+ exchanger NCLX exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction. However, the physiological relevance of the mitochondria-SR Ca2+ interaction has remained unsolved. Furthermore, although mitochondrial Ca2+ has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases, the contribution of the Ca2+-dependent regulatory mechanisms to cellular functions under physiological conditions has been controversial. In this study, we constructed a new integrated model of human ventricular myocyte with excitation-contraction-energetics coupling and investigated systematically the contribution of mitochondria-SR Ca2+ interaction, especially focusing on cardiac energetics during dynamic workload transitions in exercise. Simulation analyses revealed that the spatial coupling of mitochondria and SR, particularly via mitochondrial Ca2+ uniport activity-RyR, was the primary determinant of mitochondrial Ca2+ concentration, and that the Ca2+-dependent regulatory mechanism facilitated mitochondrial NADH recovery during exercise and contributed to the stability of NADH in the workload transition by about 40%, while oxygen consumption rate and cytoplasmic ATP level were not influenced. We concluded that the mitochondria-SR Ca2+ interaction, created via the uneven distribution of Ca2+ handling proteins, optimizes the contribution of the mitochondrial Ca2+-dependent regulatory mechanism to stabilizing NADH during exercise. KEY POINTS: The mitochondrial Ca2+ uniporter protein MCU and the Na+-Ca2+ exchanger protein NCLX are reported to exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction in cardiomyocytes. Mitochondrial Ca2+ (Ca2+ mit) has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases. Here we constructed an integrated model of a human ventricular myocyte with excitation-contraction-energetics coupling and investigated the role of the mitochondria-SR Ca2+ interaction in cardiac energetics during exercise. Simulation analyses revealed that the spatial coupling particularly via mitochondrial Ca2+ uniport activity-RyR is the primary determinant of Ca2+ mit concentration, and that the activation of NADH-producing dehydrogenases by Ca2+ mit contributes to NADH stability during exercise. The mitochondria-SR Ca2+ interaction optimizes the contribution of Ca2+ mit to the activation of NADH-producing dehydrogenases.

The highly conserved region within exonuclease III-like in PML-I regulates the cytoplasmic localization of PML-NBs

The sub-nuclear protein structure PML-NB regulates a wide range of important cellular functions, while its abnormal cytoplasmic localization may have pathological consequences. However, the nature of this aberrant localization remains poorly understood. In this study, we unveil that PML-I, the most conserved and abundant structural protein of PML-NB, possesses potent cytoplasmic targeting ability within the N-terminal half of the exonuclease III-like domain encoded by its unique exon 9, independent of the known nuclear localization signal. Fusion of this region to PML-VI can relocate PML-VI from the nucleus to the cytosol. Structural and deletion analysis revealed that the cytoplasmic targeting ability of this domain was restrained by the sequences encoded by exon 8a and the 3’ portion of exon 9 in PML-I. Deletion of either of these regions relocates PML-I to the cytosol. Furthermore, we observed a potential interaction between the ER-localized TREX1 and the cytoplasmic-located PML-I mutants. Our results suggest that perturbation of the EXO-like domain of PML-I may represent an important mode to translocate PMLs from the nucleus to the cytosol, thereby interfering with the normal nuclear functions of PML-NBs.

CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription.

Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identified a total of 39 functional enhancers for four dosage-sensitive genes, APP , FMR1 , MECP2 , and SIN3A , using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. We found that enhancer annotation provides potential pathological insights into disease-associated copy number variants. More importantly, we discovered that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription is stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A , and potentially other dosage-sensitive genes.

MiR-5195-3p predicts clinical prognosis and represses colorectal cancer progression by targeting TLR4/MyD88 signaling

BackgroundRecent studies have highlighted the role of miR-5195-3p in suppressing cell growth in various cancers. However, the specific functional impact of miR-5195-3p in colorectal cancer (CRC) remain to be fully clarified. The importance of miR-5195-3p in CRC was evaluated, aiming to uncover its underlying molecular mechanism and identify it as a potential therapeutic target for CRC.ResultsOur research has shown that miR-5195-3p is markedly under-expressed in CRC tissues and cell cultures, with its reduced presence associated with a higher TNM stage, lymphatic invasion, and unfavorable survival outcome. Ectopic miR-5195-3p expression curtailed proliferation, migration, and invasion of SW1116 and HT29 cells. Additionally, we discovered that miR-5195-3p directly targets and negatively influences Toll-like receptor 4 (TLR4) in CRC cells. Moreover, an inverse relationship was noted between miR-5195-3p and TLR4 expression in CRC tissue samples. Notably, restoring TLR4 expression counteracted miR-5195-3p’s suppressive impact on cell growth, motility, invasiveness, epithelial-mesenchymal transition (EMT), and the TLR4/MyD88 signaling pathway in SW1116 and HT29 cells.ConclusionsMiR-5195-3p suppresses the CRC cellular functions through the downregulation of TLR4/MyD88 signaling, indicating that targeting miR-5195-3p might offer a viable therapeutic strategy for CRC.