Cell Assay Research Articles

Lymphocyte antigen 6 family member E suppresses apoptosis and promotes pancreatic cancer growth and migration via Wnt/β-catenin pathway activation

Pancreatic cancer (PC) is the primary cause of cancer-related mortality. Due to the absence of reliable biomarkers for predicting prognosis or guiding treatment, there is an urgent need for molecular studies on PC. Lymphocyte antigen 6 family member E (LY6E) is implicated in uncontrolled cell growth across various cancers. However, the precise mechanism of LY6E in PC remains unclear. Here, we conducted comprehensive bioinformatic analyses using online tools and R- × 64–4.1.1, complemented by experimental validation through Western blotting, immunohistochemistry, immunosorbent assays, flow cytometry, cell assays, and animal models. Our findings reveal significantly elevated expression of LY6E in PC, correlating with poor prognosis. LY6E knockdown inhibited proliferation, invasion, and migration of PC cells, while enhancing apoptosis evidenced by increased cleaved caspase 3 levels and alterations in the Bcl-2/Bax ratio. Conversely, LY6E overexpression promoted PC cell proliferation and migration, and inhibited apoptosis. Mechanistically, LY6E downregulation suppressed the Wnt/β-catenin signaling pathway. In vivo studies demonstrated that LY6E suppression attenuated tumor growth in murine models. Additionally, LY6E suppression resulted in reduced tumor growth in mice. In conclusion, our study confirms the significant role of LY6E in the progression of PC. LY6E, serving as an independent prognostic indicator, has the potential to serve as a valuable biomarker for PC to inform treatment strategies.

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis

Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation–immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

Three-Dimensionally Printed Microsystems to Facilitate Flow-Based Study of Cells from Neurovascular Barriers of the Retina.

Rapid prototyping has produced accessible manufacturing methods that offer faster and more cost-effective ways to develop microscale systems for cellular testing. Commercial 3D printers are now increasingly adapted for soft lithography, where elastomers are used in tandem with 3D-printed substrates to produce in vitro cell assays. Newfound abilities to prototype cellular systems have begun to expand fundamental bioengineering research in the visual system to complement tissue engineering studies reliant upon complex microtechnology. This project used 3D printing to develop elastomeric devices that examined the responses of retinal cells to flow. Our experiments fabricated molds for elastomers using metal milling, resin stereolithography, and fused deposition modeling via plastic 3D printing. The systems were connected to flow pumps to simulate different flow conditions and examined phenotypic responses of endothelial and neural cells significant to neurovascular barriers of the retina. The results indicated that microdevices produced using 3D-printed methods demonstrated differences in cell survival and morphology in response to external flow that are significant to barrier tissue function. Modern 3D printing technology shows great potential for the rapid production and testing of retinal cell responses that will contribute to both our understanding of fundamental cell response and the development of new therapies. Future studies will incorporate varied flow stimuli as well as different extracellular matrices and expanded subsets of retinal cells.

Renin and (pro)renin receptors induce vascular smooth muscle cell proliferation and neointimal hyperplasia by activating oxidative stress and inflammation.

Introduction: Renin and prorenin promote the proliferation of vascular smooth muscle cells (VSMCs) through the (pro)renin receptor, or (P)RR, to promote restenosis occurrence. This study aimed to explore whether prorenin promoted the proliferation of VSMCs in a (P)RR-mediated Ang II-independent manner. Methods: Losartan and PD123319 were used to block the interaction between (P)RR and angiotensin in vitro. Cells were treated with renin, platelet-derived growth factor (PDGF), or RNAi-(P)RR, either jointly or individually. Cell proliferation was measured via Cell Counting Kit-8 (CCK-8) and flow cytometry methods; moreover, real-time polymerase chain reaction (RT-PCR) and Western blot (WB) assays were used to detect the expression of cyclin D1, proliferating cell nuclear antigen (PCNA), (P)RR, NOX1, and phosphatidylinositol 3-kinase (PI3K)/AKT signaling proteins. Immunofluorescence staining was conducted to measure the expression of (P)RR, and the levels of renin, PDGF-BB, inflammatory factors, and oxidative stress were determined by using enzyme-linked immunosorbent assay (ELISA). Moreover, a balloon catheter was used to enlarge the carotid artery of the Sprague Dawley rats. PRO20 was applied to identify angiotensin II (Ang II). The hematoxylin and eosin, RT-PCR, and WB results validated the cell assay results. Results: Renin promoted the proliferation of rat VSMCs by enhancing cell viability and cell cycle protein expression when Ang II was blocked, but silencing (P)RR inhibited this effect. Furthermore, renin enhanced NOX1-mediated oxidative stress and inflammation by activating the extracellular signal-regulated kinase 1/2 (ERK1/2)-AKT pathway in vitro. Similarly, the inhibition of (P)RR resulted in the opposite phenomenon. Importantly, the inhibition of (P)RR inhibited neointimal hyperplasia in vivo after common carotid artery injury by restraining NOX1-mediated oxidative stress through the downregulation of the ERK1/2-AKT pathway. The animal study confirmed these findings. Conclusion: Renin and (P)RR induced VSMC proliferation and neointimal hyperplasia by activating oxidative stress, inflammation, and the ERK1/2-AKT pathway in an Ang II-independent manner.

Effects and risk assessment of halogenated bisphenol A derivatives on human follicle stimulating hormone receptor: An interdisciplinary study

Halogenated bisphenol A (BPA) derivatives are produced during disinfection treatment of drinking water or are synthesized as flame retardants (TCBPA or TBBPA). BPA is considered as an endocrine disruptor especially on human follicle-stimulating hormone receptor (FSHR). Using a global experimental approach, we assessed the effect of halogenated BPA derivatives on FSHR activity and estimated the risk of halogenated BPA derivatives to the reproductive health of exposed populations. For the first time, we show that FSHR binds halogenated BPA derivatives, at 10 nM, a concentration lower than those requires to modulate the activity of nuclear receptors and/or steroidogenesis enzymes. Indeed, bioluminescence assays show that FSHR response is lowered up to 42.36 % in the presence of BPA, up to 32.79 % by chlorinated BPA derivatives and up to 27.04 % by brominated BPA derivatives, at non-cytotoxic concentrations and without modification of basal receptor activity. Moreover, molecular docking, molecular dynamics simulations, and site-directed mutagenesis experiments demonstrate that the halogenated BPA derivatives bind the FSHR transmembrane domain reducing the signal transduction efficiency which lowers the cellular cAMP production and in fine disrupts the physiological effect of FSH. The potential reproductive health risk of exposed individuals was estimated by comparing urinary concentrations (through a collection of human biomonitoring data) with the lowest effective concentrations derived from in vitro cell assays. Our results suggest a potentially high concern for the risk of inhibition of the FSHR pathway. This global approach based on FSHR activity could enable the rapid characterization of the toxicity of halogenated BPA derivatives (or other compounds) and assess the associated risk of exposure to these halogenated BPA derivatives.

Fcγ-receptor-IIIA bioactivity of circulating and synovial immune complexes in rheumatoid arthritis

ObjectivePrevious technical limitations prevented the proof of Fcγ-receptor (FcγR)-activation by soluble immune complexes (sICs) in patients. FcγRIIIa (CD16) is a risk factor in rheumatoid arthritis (RA). We aimed at determining...

ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness

Backgroundp63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor β (TGFβ) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFβ activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFβ and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.MethodsSmall molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.ResultsThe sphere-forming capacity of breast cancer cells was enhanced upon TGFβ stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFβ signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFβ stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFβ effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFβ as the upstream signal for guiding ΔNp63 to the TGFβ/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFβ through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription.ConclusionsΔNp63, phosphorylated and recruited by TGFβ to the TGFβ/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

Variant-to-function mapping of late-onset Alzheimer's disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus.

Late-onset Alzheimer's disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2, ABI3, and CR1. However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1. We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD 'CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.

Anti-Allergic and Anti-Inflammatory Signaling Mechanisms of Natural Compounds/Extracts in In Vitro System of RBL-2H3 Cell: A Systematic Review.

Various extracts are tested for anti-allergic or anti-inflammatory properties on in vitro models. RBL-2H3 cells are widely used in allergic or immunological studies. FCεRI and its downstream signaling cascades, such as MAPK, NF-κB, and JAK/STAT signaling pathways, are important allergic or inflammatory signaling mechanisms in mast and basophil cells. This systematic review aims to study common signaling pathways of the anti-allergic or anti-inflammatory compounds on RBL-2H3 cells. We selected the relevant research articles published after 2015 from the PubMed, Scopus, Science Direct and Web of Science databases. The risk of bias of the studies was assessed based on the modified CONSORT checklist for in vitro studies. The cell lines, treatments, assay, primary findings, and signaling pathways on RBL-2H3 cells were extracted to synthesize the results. Thirty-eight articles were included, and FCεRI and its downstream pathways, such as Lyn, Sky, PLCγ, and MAPK, were commonly studied. Moreover, the JAK/STAT pathway was a potential signaling mechanism in RBL-2H3 cells. However, the findings based on RBL-2H3 cells needed to be tested along with human mast cells to confirm its relevance to human health. In conclusion, a single plant extract may act as an anti-inflammatory reagent in RBL-2H3 cells via multiple signaling pathways besides the MAPK signaling pathway.

Exploring the amyloid degradation potential of nanoformulated carrageenan-bridging in vitro and in vivo perspectives

Amyloids, with their β-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17–20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Positive cooperativity in synergistic activation of Wnt proteins

BackgroundWnt proteins are crucial for embryonic development, stem cell growth, and tissue regeneration. Wnt signaling pathway is activated when Wnt proteins bind to cell membrane receptors.Methods and resultsWe employed a luciferase reporter assay in HEK293STF cells to measure Wnt protein-induced signaling. We observed that Wnt3a uniquely promotes the Wnt/β-catenin pathway through positive cooperativity. Additionally, MFH-ND, a molecular mimic of Wnt ligands, markedly increased Wnt3a-induced signaling in a dose-responsive manner. This suggests that various Wnt ligands can synergistically enhance Wnt pathway activation.ConclusionsThe study suggests the likelihood of various Wnt ligands coexisting in a single signalosome on the cell membrane, providing new insights into the complexities of Wnt signaling mechanisms.

A novel molecular pathway of lipid accumulation in human hepatocytes caused by PFOA and PFOS

Exposed to ubiquitously perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) has been associated with non-alcoholic fatty liver disease (NAFLD), yet the underlying molecular mechanism remains elusive. The extrapolation of empirical studies correlating per- and polyfluoroalkyl substance (PFAS) exposure with NAFLD occurrence to real-life exposure was hindered by the limited availability of mechanistic data at environmentally relevant concentrations. Herein, a novel pathway mediating hepatocyte lipid accumulation by PFOA and PFOS at human-relevant dose (<10 μM) was identified by integrating CRISPR-Cas9 genome screening, concentration-dependent transcriptional assay in HepG2 cell and epidemiological data mining. 1) At genetic level, nudt7 showed the highest enriched potency among 569 NAFLD-related genes, and the transcription of nudt7 was significantly downregulated by PFOA and PFOS exposure (<7 μM). 2) At molecular pathway, upon exposure to ≤10−4 μM PFOA and PFOS, the downregulation of nudt7 transcriptional expression triggered the reduction of Ace-CoA hydrolase activity. 3) At cellular level, increased lipids were measured in HepG2 cells with PFOA and PFOS (<2 μM). Overall, we identified a novel mechanism mediated by transcriptional downregulation of nudt7 gene in hepatocellular lipid increase treated with PFOA and PFOS, which could potentially explain the NAFLD occurrence associated with exposure to PFASs in humans.

Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.

Flamenco (Flam) is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells, and it is required for female fertility to silence gypsy/mdg4 transposons. To determine how Flam is regulated, we used promoter-bashing reporter assays in OSS cells to uncover novel enhancer sequences within the first exons of Flam . We confirmed the enhancer sequence relevance in vivo with new Drosophila Flam deletion mutants of these regions that compromised Flam piRNA expression and lowered female fertility from activated transposons. Our proteomic analysis of proteins associated with these enhancer sequences discovered the transcription factor Traffic Jam (TJ). Tj knockdowns in OSS cells caused a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. A TJ ChIP-seq analysis from whole flies and OSS cells confirmed TJ binding exactly at the enhancer that was deleted in the new Flam mutant as well as at multiple Piwi pathway gene enhancers. Interestingly, TJ also bound the Long Terminal Repeats of transposons that had decreased expression after Tj knockdowns in OSS cells. Our study reveals the integral role TJ plays in the on-going arms race between selfish transposons and their suppression by the host Piwi pathway and the Flam piRNA cluster locus.

Comprehensive analysis of the functional impact of single nucleotide variants of human CHEK2.

Loss of function mutations in the checkpoint kinase gene CHEK2 are associated with increased risk of breast and other cancers. Most of the 3,188 unique amino acid changes that can result from non-synonymous single nucleotide variants (SNVs) of CHEK2, however, have not been tested for their impact on the function of the CHEK2-enocded protein (CHK2). One successful approach to testing the function of variants has been to test for their ability to complement mutations in the yeast ortholog of CHEK2, RAD53. This approach has been used to provide functional information on over 100 CHEK2 SNVs and the results align with functional assays in human cells and known pathogenicity. Here we tested all but two of the 4,887 possible SNVs in the CHEK2 open reading frame for their ability to complement RAD53 mutants using a high throughput technique of deep mutational scanning (DMS). Among the non-synonymous changes, 770 were damaging to protein function while 2,417 were tolerated. The results correlate well with previous structure and function data and provide a first or additional functional assay for all the variants of uncertain significance identified in clinical databases. Combined, this approach can be used to help predict the pathogenicity of CHEK2 variants of uncertain significance that are found in susceptibility screening and could be applied to other cancer risk genes.

DB-1314, a novel DLL3-targeting ADC with DNA topoisomerase I inhibitor, exhibits promising safety profile and therapeutic efficacy in preclinical small cell lung cancer models

BackgroundDelta-like ligand 3 (DLL3) is highly expressed on the cell surface of small cell lung cancer (SCLC), one of the most lethal malignancies, but minimally or not in normal tissues, making it an attractive target for SCLC. However, none of the DLL3-targeting antibody-drug conjugates (ADCs) have been approved for SCLC therapy yet. We developed DB-1314, the new anti-DLL3 ADC composed of a novel humanized anti-DLL3 monoclonal antibody (DB131401) conjugated with eight molecules of P1021 (topoisomerase I inhibitor), and described its preclinical profiles.MethodsThe binding epitope for DB131401 and Rovalpituzumab was tested by biolayer interferometry. The binding affinity and specificity of DB-1314 to DLL3 and other homologous proteins were respectively measured by surface plasmon resonance and enzyme-linked immunosorbent assay. Internalization, bystander effects, and antibody-dependent cell-mediated cytotoxicity (ADCC) were assessed by respective assay. DLL3 was quantified by antibodies bound per cell assay and immunohistochemistry. In vitro and in vivo growth inhibition studies were evaluated in SCLC cell lines, and cell line/patient-derived xenograft models. The safety profile was measured in cynomolgus monkeys.ResultsDB-1314 induces potent, durable, and dose-dependent antitumor effects in cells in vitro and in cell/patient-derived xenograft models in vivo. The killing activity of DB-1314 mechanically arises from P1021-induced DNA damage, whereby P1021 is delivered and released within tumor cells through DLL3-specific binding and efficient internalization. Bystander effects and ADCC also contribute to the antitumor activity of DB-1314. DB-1314 displays favorable pharmacokinetic and toxicokinetic profiles in rats and cynomolgus monkeys; besides, DB-1314 is well-tolerated at a dose of up to 60 mg/kg in monkeys.ConclusionsThese results suggest that DB-1314 may be a candidate ADC targeting DLL3 for the treatment of DLL3-positive SCLC, supporting further evaluation in the clinical setting.

Hepatoprotective Lignan Glycosides from the Leaves and Stems of Symplocos cochinchinensis (Lour.) S. Moore.

This study investigates Symplocos cochinchinensis (Lour.) S. Moore leaves and stems, commonly known as Symplocos, a plant indigenous to Asia renowned for its traditional use in holistic medicine. A comprehensive phytochemical analysis of S. cochinchinensis led to the isolation of two new lignans, namely symplolignans A and B (1 and 2) along with eleven known lignan glucosides: nortrachelogenin 4-O-β-D-glucopyranoside (3), nortracheloside (4), matairesinol 4-O-β-D-glucopyranoside (5), lariciresinol 4'-O-β-D-glucopyranoside (6), balanophonin 4-O-β-D-glucopyranoside (7), dehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside (8), dehydrodiconiferyl alcohol γ'-O-β-D-glucopyranoside (9), 3-(β-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofura (10), and pinoresinol 4'-O-β-D-glucopyranoside (11). Their chemical structures were elucidated using 1D- and 2D-NMR, mass spectrometry, and their spectroscopic data were compared with those reported in literatures. Furthermore, all compounds were evaluated for their hepatoprotective effects using the Resazurin reduction assay in HepG2 hepatocellular carcinoma cells. Compounds 1, 5, 7, and 8 exhibited notable hepatoprotective efficacy, with cell viability ranging from 105.0±2.6 to 109.2±3.3 at a concentration of 10 μM. This research highlights the therapeutic potential of these compounds and enhanced to the understanding of lignans and neolignans in liver cell proliferation.

Tripodal BODIPY-Tagged and Functional Molecular Probes: Synthesis, Computational Investigations and Explorations by Multiphoton Fluorescence Lifetime Imaging Microscopy.

A range of novel BODIPY derivatives with a tripodal aromatic core was synthesized and characterized spectroscopically. These new fluorophores showed promising features as probes for in vitro assays in live cells and offer strategic routes for further functionalization towards hybrid nanomaterials. Incorporation of biotin tags facilitated proof-of-concept access to targeted bioconjugates as molecular probes. Computational explorations using DFT and TD-DFT calculations identified the most stable tripodal linker conformations and predicted their absorption and emission behavior. The uptake and speciation of these molecules in living prostate cancer cells was imaged by single- and two-photon excitation techniques coupled with two-photon fluorescence lifetime imaging (2P FLIM).

Investigation of Cytotoxic Effects and Antimicrobial Activities of Light-cured and Self-cured Universal Adhesive Systems

Objective: This study aimed to compare the cytotoxicity and antimicrobial activity of a light-cured adhesive system and a self-cured adhesive system from the same company. Materials and Methods: A Tokuyama BOND force II (Light-cured) adhesive system (TF2B) and a Tokuyama Universal Bond (Self-cured) adhesive system (TUB) were selected for the study. The cytotoxicity evaluation of these two systems on cell cultures was performed using MTT assay and Agar Diffusion assay in L929 fibroblast cells. Disk diffusion method and liquid microdilution (MIC) method were used to evaluate their antimicrobial activity. The experiments were performed on 6 pathogenic bacteria and 1 yeast fungus. Results: According to MTT test results, both adhesive systems have no significant toxic effect on healthy cells (L929). However, when TUB and TF2B were compared with each other, it was found that TF2B had almost no toxic effect. In the agar diffusion test, when the two bonds were compared with each other, a weak color lightening was observed only around the first concentration of TUB. No visible melting was detected in other concentrations of TUB and TF2B. Both adhesive systems failed to reach MIC values effectively on the test microorganisms. Since the results were far above the MIC values of the reference antibiotics, it was determined that they did not have antimicrobial effects. Disk diffusion results similarly showed that both bonds did not form an inhibition zone on the test microorganisms. Conclusions: In dentistry, cytotoxic effects of universal adhesive systems on living cells can be observed. Self-cured and Light-cured adhesive systems did not show toxic effects on L929 cells. In addition, antimicrobial effects on test microorganisms were not detected. The cytotoxicity of the materials can be tested on different cells.

GelMA loaded with platelet lysate promotes skin regeneration and angiogenesis in pressure ulcers by activating STAT3

Pressure ulcers (PU) are caused by persistent long-term pressure, which compromises the integrity of the epidermis, dermis, and subcutaneous adipose tissue layer by layer, making it difficult to heal. Platelet products such as platelet lysate (PL) can promote tissue regeneration by secreting numerous growth factors based on clinical studies on skin wound healing. However, the components of PL are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel that has lately emerged as a promising material for tissue engineering and regenerative medicine. The PL liquid was extracted, flow cytometrically detected for CD41a markers, and evenly dispersed in the GelMA hydrogel to produce a surplus growth factor hydrogel system (PL@GM). The microstructure of the hydrogel system was observed under a scanning electron microscope, and its sustained release efficiency and biological safety were tested in vitro. Cell viability and migration of human dermal fibroblasts, and tube formation assays of human umbilical vein endothelial cells were applied to evaluate the ability of PL to promote wound healing and regeneration in vitro. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to elucidate the skin regeneration mechanism of PL. We verified PL’s therapeutic effectiveness and histological analysis on the PU model. PL promoted cell viability, migration, wound healing and angiogenesis in vitro. Real-time PCR and western blot indicated PL suppressed inflammation and promoted collagen I synthesis by activating STAT3. PL@GM hydrogel system demonstrated optimal biocompatibility and favorable effects on essential cells for wound healing. PL@GM also significantly stimulated PU healing, skin regeneration, and the formation of subcutaneous collagen and blood vessels. PL@GM could accelerate PU healing by promoting fibroblasts to migrate and secrete collagen and endothelial cells to vascularize. PL@GM promises to be an effective and convenient treatment modality for PU, like chronic wound treatment.

DNA damage in workers exposed to mineral oils

Mineral oils, untreated or mildly treated, have been classified in group 1 as a potential source of cancer by the International Agency for Research on Cancer (IARC). Although numerous studies have implicated metalworking fluids (MWFs) as human carcinogens, toxicology data regarding the mechanism of carcinogenicity are limited. This study is intended to examine the systemic effects of machining workers’ exposure to MWFs. The potential toxicity of mineral oils was investigated in 65 lathe workers compared to controls (66 men). The occupational exposure was measured by the National Institute for Occupational Safety and Health (NIOSH) 5026. The DNA damage has been examined by the comet assay method. According to the field assessments, the time-weighted average (TWA) exposure to mineral oil mist was 7.67 ± 3.21 mg/m3. A comet assay of peripheral blood cells showed that tail length (TL) and olive moment (OM) were significantly higher in the exposed group (p < 0.05). A multiple logistic regression analysis revealed that, within subjects with over 10 years of exposure, the odds ratio of worker with high TL, percent of DNA in tail, OM, and tail moment (TM) were 1.68, 1.41, 1.71, and 2.71, respectively. DNA strand break in exposed workers was associated with higher exposure time in years. Mineral oil toxicity could be altered in the presence of by-products and impurities. For a better understanding of genotoxicity, further studies are required.