Regulation Of Cell Differentiation Research Articles

Tyrosine Hydroxylase-positive Nucleus Accumbens Neurons Influence Delay Discounting in a Mouse T-maze Task.

Delay discounting (DD) is a phenomenon where individuals devalue a reward associated with a temporal delay, with the rate of devaluation being representative of impulsive-like behavior. Here we first sought to develop and validate a mouse DD task to study brain circuits involved in DD decision-making within short developmental time windows, given widespread evidence of developmental regulation of impulse control and risk-taking. We optimized a T-maze DD task for mice that enables training and DD trials within two weeks. Mice learned to choose between a large and a small reward located at opposite arms of a T-maze. Once training criteria were met, mice underwent DD whereby the large reward choice was associated with a temporal delay. Task validation showed that adolescent C57BL/6J mice display increased preference for the small reward upon a temporal delay, confirming increased impulsivity compared to adults. We next used this DD task to explore the neural basis of decision-making. We used tyrosine hydroxylase transgenic mice (TH-Cre) to target TH-positive neurons in the nucleus accumbens (NAc) and ventral tegmental area (VTA) with Cre-dependent excitatory or inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Inhibition of transduced neurons in the NAc decreased preference for the small but immediate reward during DD. Inhibition of TH+ neurons in the ventral tegmental area (VTA) did not affect impulsive choice in this DD task. These results uncover a novel role for NAc TH-positive neurons in DD behavior and expand the repertoire of behavioral tasks available for studying decision-making across the lifespan.Significance Statement Delay discounting (DD) tasks are used in rodents to study impulsive choice, whereby subjects display a preference for an immediate, smaller reward when access to a larger reward is contingent on a temporal delay. Research implicates the nucleus accumbens (NAc) brain region in impulsive behavior, with recent evidence of specialization among NAc neuronal subtypes in impulsive choice. Here we interrogated the neural requirements of impulsive choice in mice. We found that inhibition of a subset of NAc neurons expressing tyrosine hydroxylase decreases impulsive choice. We also saw increased impulsive choice in adolescent mice compared to adults, consistent with reported developmental changes in impulsivity. Together, our data identify cell-specific NAc regulation of impulsive choice with important implications for neurodevelopment.

Iodine activates NLRP3 inflammasomesin PBMCs of patients with autoimmune thyroiditis, and regulates Th1 and Th17 cell differentiation.

Inflammasomes are associated with various autoimmune diseases. Herein, we aimed to study the occurrence of inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients with autoimmune thyroiditis (AIT), and the relationship between their abundance and the inflammatory response index of AIT. Furthermore, we examined the effect of iodine on inflammasomes containing NLR family pyrin domain containing 3 (NLRP3) and inflammasome activation of helper T cell (Th) differentiation regulation in cultured PBMCs.

Rbm24-mediated post-transcriptional regulation of skeletal and cardiac muscle development, function and regeneration.

RNA-binding proteins are critically involved in the post-transcriptional control of gene expression during embryonic development and in adult life, contributing to regulating cell differentiation and maintaining tissue homeostasis. Compared to the relatively well documented functions of transcription factors, the regulatory roles of RNA-binding proteins in muscle development and function remain largely elusive. However, deficiency of many RNA-binding proteins has been associated with muscular defects, neuromuscular disorders and heart diseases, such as myotonic dystrophy, amyotrophic lateral sclerosis, and cardiomyopathy. Rbm24 is highly conserved among vertebrates and is one of the best characterized RNA-binding proteins with crucial implication in the myogenic and cardiomyogenic programs. It presents the distinctive particularity of displaying highly restricted expression in both skeletal and cardiac muscles, with changes in subcellular localization during the process of differentiation. Functional analyses using different vertebrate models have clearly demonstrated its requirement for skeletal muscle differentiation and regeneration as well as for myocardium organization and cardiac function, by regulating the expression of both common and distinct target genes in these tissues. The challenge remains to decipher the dynamic feature of post-transcriptional circuits regulated by Rbm24 during skeletal myogenesis, cardiomyogenesis, and muscle repair. This review discusses current understanding of its function in striated muscles and its possible implication in human disease, with the aim of identifying research gaps for future investigation.

Developmental Stage and Cellular Context Determine Oncogenic and Molecular Outcomes of Ezh2Y641F Mutation in Hematopoiesis.

Mutations in the histone methyltransferase EZH2, particularly the Y641 hotspot mutation, have been implicated in hematologic malignancies, yet the effect of timing and cellular context on their oncogenic potential has remained unknown. In this study, we utilized a conditional allele with tissue-specific Cre drivers to investigate the effects of Ezh2Y641F mutations at various stages of development, with a focus on the hematopoietic system. We found that ubiquitous heterozygous Ezh2Y641F expression at birth, or conditional expression in hematopoietic or mesenchymal stem cells, led to decreased survival due to hematopoietic defects and bone marrow failure, with no evidence of malignancy. In contrast, Ezh2Y641F expression in committed B cells drives lymphoma formation, highlighting the lineage-specific oncogenic activity of the mutation. Transcriptomic analysis of B cell progenitors revealed key pathway alterations between Cre models such as altered IL2-Stat5 signaling pathway, differential expression of E2F targets, and altered GTPase pathway expression driven by upregulation of Guanylate Binding Proteins (GBPs) in Mx1-Cre Ezh2Y641F pro-B cells. We further found that the GBP locus is regulated by Ezh2-mediated H3K27me3, it is associated with poorer survival in Acute Myeloid Leukemia patients and has variable effects on apoptosis in human lymphoma and leukemia cell lines. These findings suggest that the Ezh2Y641F mutation may alter immune regulatory pathways, cell differentiation and apoptosis, with potential implications for disease progression. Our results highlight the critical role of mutation timing and cellular context in EZH2-driven hematopoietic disease, resulting in distinct downstream changes that shape the oncogenic impact of EZH2.

Genomes of two invasive Adelges species (hemlock woolly adelgid and pineapple gall adelgid) enable characterization of nicotinic acetylcholine receptors.

Two invasive hemipteran adelgids cause widespread damage to North American conifers. Adelges tsugae (the hemlock woolly adelgid) has decimated Tsuga canadensis and Tsuga caroliniana (the Eastern and Carolina hemlocks, respectively). A. tsugae was introduced from East Asia and reproduces parthenogenetically in North America, where it can kill trees rapidly. A. abietis, introduced from Europe, makes pineapple galls on several North American spruce species, and weakens trees, increasing their susceptibility to other stresses. Broad-spectrum insecticides that are often used to control adelgid populations can have off-target impacts on beneficial insects and the development of more selective chemical treatments could improve control methods and minimize ecological damage. Whole genome sequencing was performed on both species to aid in development of targeted pest control solutions and improve species conservation. The assembled A. tsugae and A. abietis genomes are 231.71 Mbp and 290.39 Mbp, respectively, each consisting of nine chromosomes and both genomes are over 96% complete based on BUSCO assessment. Genome annotation identified 11,424 and 14,118 protein-coding genes in A. tsugae and A. abietis, respectively. Comparative analysis across 29 Hemipteran species and 14 arthropod outgroups identified 31,666 putative gene families. Gene family expansions in A. abietis included ABC transporters and carboxypeptidases involved in carbohydrate metabolism, while both species showed contractions in core histone families and oxidoreductase pathways. Gene family expansions in A. tsugae highlighted families associated with the regulation of cell differentiation and development (survival motor protein, SMN; juvenile hormone acid methyltransferase JHAMT) as well as those that may be involved in the suppression of plant immunity (clip domain serine protease-D, CLIPD; Endoplasmic reticulum aminopeptidase 1, ERAP1). Among the analyzed gene families, Nicotinic acetylcholine receptors (nAChRs) maintained consistent copy numbers and structural features across species, a finding particularly relevant given their role as targets for current forestry management insecticides. Detailed phylogenetic analysis of nAChR subunits across adelgids and other ecologically important insects revealed remarkable conservation in both sequence composition and predicted structural features, providing crucial insights for the development of more selective pest control strategies.

Ezh2 Delays Activation of Differentiation Genes During Normal Cerebellar Granule Neuron Development and in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumour in children. The Sonic Hedgehog (SHH)-medulloblastoma subtype arises from the cerebellar granule neuron lineage. Terminally differentiated neurons are incapable of undergoing further cell division, so an effective treatment for this tumour could be to force neuronal differentiation. Differentiation therapy provides a potential alternative for patients with medulloblastoma who harbor mutations that impair cell death pathways (TP53), which is associated a with high mortality. To this end, our goal was to explore epigenetic regulation of cerebellar granule neuron differentiation in medulloblastoma cells. Key regulators were discovered using chromatin immunoprecipitation with high-throughput sequencing. DNA-bound protein and chromatin protein modifications were investigated across all genes. We discovered that Ezh2-mediated tri-methylation of the H3 histone (H3K27me3), occurred on more than half of the 787 genes whose transcription normally increases as granule neurons terminally differentiate. Conditional knockout of Ezh2 led to early initiation of differentiation in granule neuron precursors (GNPs), but only after cell cycle exit had occurred. Similarly, in MB cells, neuronal differentiation could be induced by preventing H3K27me3 modifications using an Ezh2 inhibitor (UNC1999), but only when UNC1999 was combined with forced cell cycle exit driven by a CDK4/6 inhibitor (Palbociclib). Ezh2 emerges as a powerful restraint upon post-mitotic differentiation during normal GNP development and combination of Ezh2 inhibition with cell cycle exit leads to MB cell differentiation.

Exploration of the molecular mechanism of Wufu Yin in the treatment of knee osteoarthritis based on network pharmacology and experimental validation

Wufu Yin (WFY) exhibits significant clinical effectiveness in knee osteoarthritis (KOA) treatment, yet its therapeutic mechanisms are still unclear. This study aimed to explore the active ingredients and potential mechanism of WFY in the treatment of KOA. The network pharmacology-based approach was adopted to investigate the underlying mechanism of WFY in treating KOA. Molecular docking analysis was performed using Auto Vina software. An in vitro model of KOA inflammation was established by inducing chondrocyte cultures with interleukin-1 beta (IL-1β). Cell viability was quantified through the cell counting kit-8 assay, inflammatory cytokine levels were measured via ELISA, and protein expressions were assessed by Western blot analysis. A total of 225 active ingredients and 265 targets of WFY were identified, of which 88 were identified as potential targets against KOA. Enrichment analysis showed that these targets were associated with oxidative stress, cell proliferation and apoptosis, and inflammatory response, and were involved in the regulation of Th17 cell differentiation, IL-17 signaling pathway, tumor necrosis factor signaling pathway, and other signaling pathways. Topology analysis showed that PTGS2, NOS2, ESR11, PPARG, and MAPK14 had higher degree values and were key targets of WFY in the treatment of KOA. Molecular docking analysis showed that these key targets and active ingredients had low binding energies, indicating that they had potential binding activity. Furthermore, IL-1β-induced elevation of inflammatory cytokines, PTGS2 protein expression, and phosphorylated p38/p38 ratios in chondrocytes were significantly attenuated upon WFY intervention. Our study systematically elucidated the pharmacological basis and molecular mechanism underlying WFY’s therapeutic effects in KOA, substantiating its ability to suppress inflammation and regulate PTGS2 expression and p38 phosphorylation.

The CCN Family of Proteins: A Critical Approach to the Multi-Modular Structure of the CCN Domains

The CCN family of proteins is composed of six members (CCN1-CCN6) sharing a tetra-modular organization and a striking conservation of their primary structure. The CCN acronym was originally assigned in 1993 by P. Bork to three newly discovered factors (originally called CTGF, CYR61, and NOV), which he proposed to constitute a new family of proteins on the basis of their common physical features. Six years later, three other proteins (Wisp1-3), sharing the same tetramodular organization, joined the family (figure 1). The HUGO-recognized acronyms for the CCN proteins were officialized in 2018[1]. The CCN family turned out to contain positive and negative regulators of cell proliferation and differentiation, with pro- and anti-tumorigenic activities. A significant amount of work has been performed to identify the participation of the constitutive modules in these biological features. The aim of this review is to briefly examine the potential roles assigned to the constitutive modules of CCN proteins and propose a critical view of the structural basis for their interactions and functions.

YAP and ECM Stiffness: Key Drivers of Adipocyte Differentiation and Lipid Accumulation.

ECM stiffness significantly influences the differentiation of adipose-derived stem cells (ADSCs), with YAP-a key transcription factor in the Hippo signaling pathway-playing a pivotal role. This study investigates the effects of ECM stiffness on ADSC differentiation and its relationship with YAP signaling. Various hydrogel concentrations were employed to simulate different levels of ECM stiffness, and their impact on ADSC differentiation was assessed through material properties, adipocyte-specific gene expression, lipid droplet staining, YAP localization, and protein levels. Our results demonstrated that increasing hydrogel stiffness enhanced adipocyte differentiation in a gradient manner. Notably, inhibiting YAP signaling further increased lipid droplet accumulation, suggesting that ECM stiffness influences adipogenesis by modulating YAP signaling and its cytoplasmic phosphorylation. This study elucidates the molecular mechanisms underlying ECM stiffness-dependent lipid deposition, highlighting YAP's regulatory role in adipogenesis. These findings provide valuable insights into the regulation of cell differentiation and have important implications for tissue engineering and obesity treatment strategies.

The heterogeneity of NOTCH1 to tumor immune infiltration in pan-cancer

NOTCH1 signaling, a vital regulator of cell proliferation and differentiation, is widely involved in the occurrence and development of malignant tumors. Pharmacological regulation of NOTCH1 is promising in tumor immunotherapy, whereas the effective rate of existing therapies remains low. NOTCH1 functions, as a cancer suppressor or a cancer promoter in different cancers, is engaged in the crosstalk between the immune microenvironment and cancer cells, posing a major challenge to immunotherapy. Therefore, a comprehensive view of the overall situation of NOTCH1-associated immune infiltration in pan-cancer should be built. The relation between NOTCH1 and immune infiltration was initially investigated in this paper. In this study, the data originated from the Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA) databases were input into multiple online bioinformatic tools to study the characteristics of NOTCH1 in pan-cancer. We found that there was obvious heterogeneity in the NOTCH1-associated tumor immune infiltration in pan-cancer. In accordance with the heterogeneity, pan-cancer mainly fell into two categories, i.e., cancers that NOTCH1 promoted immune infiltration (termed hot tumors) and NOTCH1 inhibited immune infiltration (termed cold tumors). We further analyzed the changes of immune infiltration in pan-carcinoma species from the perspectives of NOTCH1 expression, mutation, gene function, tumor metastasis and drugs. NOTCH1 expression was significantly up-regulated in cold tumors but down-regulated in hot tumors. The Gene ontology (GO) enrichment analysis of NOTCH1 with the two categories placed stress on angiogenesis and protein dealkylation, respectively. Further, the gene sets of angiogenesis facilitated immune infiltration, whereas the gene sets of protein dealkylation hindered immune infiltration. The tsRNA associated with NOTCH1 is a type of angiogenin that potentially exerts a significant influence on angiogenesis. We have conducted a meticulous analysis of the function of this tsRNA. NOTCH1 was conducive to cancer-associated fibroblasts (CAFs) immune infiltration, while the metastatic process was more dependent on the differentiation and angiogenesis function of NOTCH1. Accordingly, the heterogeneity of NOTCH1 in immune infiltration was extensively analyzed in this study based on the pan-cancer study, which can contribute to the formulation of specific immunotherapy strategies.

M5C related-regulator-mediated methylation modification patterns and prognostic significance in breast cancer

5-Methylcytosine (m5C) is closely associated with cancer. However, the role of m5C in breast cancer(BC)remains unclear. This study combined single-cell RNA sequencing (scRNA-Seq) and transcriptomics datasets to screen m5C regulators associated with BC progression and analyze their clinical values. Firstly, This study elucidates the mechanisms of the m5C landscape and the specific roles of m5C regulators in BC patients. we found that the dysregulation of m5C regulators with m5Cscore play the essential role of the carcinogenesis and progression in epithelial cells and myeloid cells of BC at single cell level. External validation was conducted using an independent scRNA-Seq datasets. Then, three distinct m5C modification patterns were identified by transcriptomics datasets. Based on the m5C differentially expressed regulators, the m5Cscore was constructed, and used to divide patients with BC into high and low m5Cscore groups. Patients with a high m5Cscore had more abundant immune cell infiltration, stronger antitumor immunity, and better prognoses. Finally, Quantitative real-time (PCR) and immunohistochemistry were used for the in vitro experimental validation, which had extensive prognostic value. In this study, we aimed to assess the expression of m5C regulators involved in BC and investigate their correlation with the tumor microenvironment, clinicopathological characteristics, and prognosis of BC. The m5C regulators could be used to effectively assess the cell specific regulation prognosis of patients with BC and develop more effective immunotherapy strategies.

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

Characterization of Single-Cell Cis-regulatory Elements Informs Implications for Cell Differentiation.

Cis-regulatory elements govern the specific patterns and dynamics of gene expression in cells during development, which are the fundamental mechanisms behind cell differentiation. However, the genomic characteristics of single-cell cis-regulatory elements closely linked to cell differentiation during development remain unclear. To explore this, we systematically analyzed ∼250,000 putative single-cell cis-regulatory elements obtained from snATAC-seq analysis of the developing mouse cerebellum. We found that over 80% of these single-cell cis-regulatory elements show pleiotropic effects, being active in 2 or more cell types. The pleiotropic degrees of proximal and distal single-cell cis-regulatory elements are positively correlated with the density and diversity of transcription factor binding motifs and GC content. There is a negative correlation between the pleiotropic degrees of single-cell cis-regulatory elements and their distances to the nearest transcription start sites, and proximal single-cell cis-regulatory elements display higher relevance strengths than distal ones. Furthermore, both proximal and distal single-cell cis-regulatory elements related to cell differentiation exhibit enhanced sequence-level evolutionary conservation, increased density and diversity of transcription factor binding motifs, elevated GC content, and greater distances from their nearest genes. Together, our findings reveal the general genomic characteristics of putative single-cell cis-regulatory elements and provide insights into the genomic and evolutionary mechanisms by which single-cell cis-regulatory elements regulate cell differentiation during development.

GABA in early brain development: A dual role review.

This comprehensive review examines the multifaceted roles of gamma-aminobutyric acid (GABA) in early brain development. GABA, traditionally recognized for its inhibitory functions in the mature brain, also exhibits excitatory effects during early neural development. This article explores the mechanisms behind GABA's dual roles, detailing its impact on the properties of the immature brain, the mechanisms of GABA-mediated excitation, the role of GABA-mediated presynaptic inhibition, the trophic actions of GABA during early development, GABA regulation of neurite growth and GABA-mediated cell differentiation in the immature brain. Emphasizing recent research findings, the review highlights the significance of GABAergic signalling in shaping the developing brain and its potential implications for understanding neurodevelopmental disorders.

The sodium/ascorbic acid co-transporter SVCT2 distributes in a striated membrane-enriched domain at the M-band level in slow-twitch skeletal muscle fibers

BackgroundVitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited.ResultsIn this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern.ConclusionsOur results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.

Judy Lieberman: Stay curious and excited about science.

Judy Lieberman is a professor of pediatrics and adjunct professor of genetics at Harvard Medical School and an endowed chair in cellular and molecular medicine. Her lab studies cytotoxic T lymphocytes (CTL), key cells in the immune defense against viral infection and cancer, as well as molecular pathways activated by the granzymes, and how RNA interference (RNAi) regulates cell differentiation in health and disease states. We spoke to Judy about advice for early career researchers, how she first become interested in cytotoxic T lymphocytes, and key people who have provided mentorship across her career.

Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction.

Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.

A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

Green Synthesis of Silver Nanoparticles with Roasted Green Tea: Applications in Alginate-Gelatin Hydrogels for Bone Regeneration.

The incorporation of silver nanoparticles (AgNPs) into alginate-gelatin (Alg-Gel) hydrogels can enhance the properties of these materials for bone regeneration applications, due to the antimicrobial properties of AgNPs and non-cytotoxic concentrations, osteoinductive properties, and regulation of stem cell proliferation and differentiation. Here, the hydrogel formulation included 2% (w/v) sodium alginate, 4 µg/mL AgNPs, and 2.5% (w/v) gelatin. AgNPs were synthesized using a 2% (w/v) aqueous extract of roasted green tea with silver nitrate. The aqueous extract of roasted green tea for AgNP synthesis was characterized using HPLC and UHPLC-ESI-QTOF-MS/MS, and antioxidant capacity was measured in Trolox equivalents (TE) from 4 to 20 nmol/well concentrations. Stem cells from human exfoliated deciduous tooth cells were used for differentiation assays including positive (SHEDs/hydrogel with AgNPs) and negative controls (hydrogel without AgNPs). FTIR was used for hydrogel chemical characterization. Statistical analysis (p < 0.05, ANOVA) confirmed significant findings. Roasted green tea extract contained caffeine (most abundant), (-)-Gallocatechin, gallic acid, and various catechins. XRD analysis revealed FCC structure, TEM showed quasispheroidal AgNPs (19.85 ± 3 nm), and UV-Vis indicated a plasmon surface of 418 nm. This integration of nanotechnology and biomaterials shows promise for addressing bone tissue loss in clinical and surgical settings.

Genome-Wide CRISPR Screen Identifies Genes Involved in Metastasis of Pancreatic Ductal Adenocarcinoma.

Background/Objectives: Early and aggressive metastasis is a major feature of pancreatic ductal adenocarcinoma. Understanding the processes underlying metastasis is crucial for making a difference to disease outcome. Towards these ends, we looked in a comprehensive manner for genes that are metastasis-specific. Methods: A genome-wide CRISPR-Cas9 gene knockout screen with 259,900 single guide RNA constructs was performed on pancreatic cancer cell lines with very high or very low metastatic capacity, respectively. Functional aspects of some of the identified genes were analysed in vitro. The injection of tumour cells with or without a gene knockout into mice was used to confirm the effect on metastasis. Results: The knockout of 590 genes-and, with higher analysis stringency, 67 genes-affected the viability of metastatic cells substantially, while these genes were not vital to non-metastasizing cells. Further evaluations identified different molecular processes related to this observation. One of the genes was MYBL2, encoding for a well-known transcription factor involved in the regulation of cell survival, proliferation, and differentiation in cancer tissues. In our metastasis-focussed study, no novel functional activity was detected for MYBL2, however. Instead, a metastasis-specific transformation of its genetic interaction with FOXM1 was observed. The interaction was synergistic in cells of low metastatic capacity, while there was a strong switch to a buffering mode in metastatic cells. In vivo analyses confirmed the strong effect of MYBL2 on metastasis. Conclusions: The genes found to be critical for the viability of metastatic cells form a basis for further investigations of the processes responsible for triggering and driving metastasis. As shown for MYBL2, unexpected processes of regulating metastasis might also be involved.