Probe Kit Research Articles

Integrating datasets from herbarium specimens and images to treat a Neotropical myrtle species complex.

Herbaria are the most important source of information for plant taxonomic work. Resources and technologies available today, such as digitised collections and herbarium DNA sequencing, can help accelerate taxonomic decisions in challenging plant groups. Here we employ an integrative methodology relying exclusively on herbarium specimens to investigate species boundaries in the Neotropical Myrcia neoobscura complex (Myrtaceae). We collected morphometric data from high-resolution images of herbarium sheets and analysed it using hierarchical clustering. We posteriorly tested the obtained morpho-groups with phylogenomics using the Angiosperms353 probe kit. We also gathered phenological and geographic information from specimen labels and built phenological histograms and ecological niche models to investigate ecological differences amongst taxa. Current circumscriptions of Myrcia arenaria, Myrcia neoglabra and Myrcia neoregeliana are confirmed in this study. Conversely, the four pieces of evidence together support Calyptranthes langsdorffii var. grandiflora, Marlierea regeliana var. parviflora and Marlierea warmingiana as separate from Myrcia marliereana, Myrcia neoriedeliana and Myrcia neoobscura, respectively, contrary to arrangements proposed by previous authors. Integrated analyses also support separation between Myrcia excoriata and two similar, undescribed taxa. Our data reveal the need for major changes in the systematics of the group, with recognition of 12 species. The successful delivery of our study aims was possible due to obtaining robust, high-quality data from museum specimens. We emphasise the importance of maintaining botanical collections physically and digitally available for taxonomic work and advocate their use to accelerate holistic taxonomic solutions of tropical species complexes. This is urgent, given the paucity of funds for fieldwork and unprecedented rates of habitat loss in the tropics.

Flexible Electromagnetic Sensor with Inkjet-Printed Silver Nanoparticles on PET Substrate for Chemical and Biomedical Applications.

For this article, a low-cost, compact, and flexible inkjet-printed electromagnetic sensor was investigated for its chemical and biomedical applications. The investigated sensor design was used to estimate variations in the concentration of chemicals (ethanol and methanol) and biochemicals (hydrocortisone-a chemical derivative of cortisol, a biomarker of stress and cardiovascular effects). The proposed design's sensitivity was further improved by carefully choosing the frequency range (0.5-4 GHz), so that the analyzed samples showed approximately linear variations in their dielectric properties. The dielectric properties were measured using a vector network analyzer (VNA) and an Agilent 85070E Dielectric Probe Kit. The sensor design had a resonant frequency at 2.2 GHz when investigated without samples, and a consistent shift in resonant frequency was observed, with variation in the concentrations of the investigated chemicals. The sensitivity of the designed sensor is decent and is comparable to its non-flexible counterparts. Furthermore, the simulation and measured results were in agreement and were comparable to similar investigated sensor prototypes based on non-flexible Rogers substrates (Rogers RO4003C) and Rogers Droid/RT 5880), demonstrating true potential for chemical, biomedical applications, and healthcare.

Truly the best of both worlds: Merging lineage‐specific and universal probe kits to maximize phylogenomic inference

AbstractPremiseHybridization capture kits are now commonly used for reduced representation approaches in genomic sequencing, with both universal and clade‐specific kits available. Here, we present a probe kit targeting 799 low‐copy genes for the plant family Annonaceae.MethodsThis new version of the kit combines the original 469 genes from the previous Annonaceae kit with 334 genes from the universal Angiosperms353 kit. We also compare the results obtained using the original Angiosperms353 kit with our custom approach using a subset of specimens. Parsimony‐informative sites and the results of maximum likelihood phylogenetic inference were assessed for combined matrices using the genera Asimina and Deeringothamnus.ResultsThe Annonaceae799 genes derived from the Angiosperms353 kit have extremely high recovery rates. Off‐target reads were also detected. When evaluating size, the proportion of on‐ and off‐target regions, and the number of parsimony‐informative sites, the genes incorporated from the Angiosperms353 panel generally outperformed the genes from the original Annonaceae probe kit.DiscussionWe demonstrated that the new sequences from the Angiosperms353 probe set are variable and relevant for future studies on species‐level phylogenomics and within‐species studies in the Annonaceae. The integration of kits also establishes a connection between projects and makes new genes available for phylogenetic and population studies.

“Making for Others”: A Creative Inquiry Into Understanding Older Men’s Motivations for Making

The do-it-yourself (DIY) and maker cultures have expanded into older adult communities and shown to bring a strong agency and well-being outcomes. Looking into well-established spaces, where older adults have engaged in making over a longer period, can provide an insight into the sustained motivations and benefits of making for older adults. Men’s sheds represent a great opportunity in this regard, as they are open, community spaces where retired, older men engage in woodworking, metalworking and other craft-based activities. We involved nine men’s shed members in a creative inquiry where a “probe kit” consisting of recycled timber and wooden materials was provided. The participants were asked to create an artifact from the kit for someone other than themselves. We interviewed them once they completed their projects and qualitatively analyzed their design process. Our findings show that older men’s involvement in making is driven by the ethos of “making for others” and can be seen across three themes: (1) creative expressions; (2) memories and social connections; and (3) practical needs. We unpack these themes in the paper and discuss factors that can play an important role when engaging with older adults.

Dihydroisotanshinone I regulates ferroptosis via PI3K/AKT pathway to enhance cisplatin sensitivity in lung adenocarcinoma.

Dihydroisotanshinone I (DT) is a kind of diterpenoid compound extracted from the dried roots of Salvia miltiorrhiza Bunge, and exhibits multiple biological activities including anti-tumor activity. Cisplatin is one of the first-line drugs for the treatment of lung adenocarcinoma (LAUD), but the drug resistance and toxicity limit its efficacy. DT is known to induce apoptosis and ferroptosis, but it is unclear whether DT can inhibit the cisplatin-resistant LAUD cells and reverse the drug resistance in LAUD. Therefore, our study intends to establish the cisplatin-resistant human LAUD cells (A549/DDP), and figure out the influence and related mechanisms of DT reversing cisplatin resistance in A549/DDP cells, so as to provide a theoretical basis for the DT as a new natural candidate for the treatment of LAUD. The establishment of A549/DDP was the continuous stimulation by exposing A549 to gradient concentrations of Cisplatin. The cell viability of A549 and A549/DDP was detected by CCK-8 kit, and the IC50 value was calculated. The morphological changes of A549 and A549/DDP cells were observed by an inverted microscope. The contents of malondialdehyde (MDA) and glutathione (GSH) in A549/DDP cells after drug treatment were detected by related kits. The levels of Fe2+, cytosolic reactive oxygen species (ROS), and lipid reactive oxygen species (lipid ROS) were detected by a fluorescence microplate reader or fluorescence cell imager according to the related fluorescent probe kit instructions. Western blot was used to detect the expressions of PI3K, phospho-PI3K, AKT, phospho-AKT, MDM2, p53, GPX4, and SLC7A11 in A549/DDP after different drug treatments. Our study demonstrated that the inhibitory effect of DT on A549 and A549/DDP cells was time-dependent and concentration-dependent, and DT and DDP had a synergistic effect on inhibiting the proliferation of A549/DDP cells. Furthermore, DT mainly induced ferroptosis in A549/DDP cells and synergized with cisplatin to promote ferroptosis in A549/DDP cells. The result of KEGG pathway analysis, molecular docking and western blot showed that DT could enhance the cisplatin sensitivity of A549/DDP by inhibiting PI3K/MDM2/P53 signaling pathway. Consequently, we concluded that DT promotes ferroptosis in cisplatin-resistant LAUD A549/DDP cells. Additionally, DT reverses cisplatin resistance by promoting ferroptosis via PI3K/MDM2/P53 pathway in A549/DDP cells.

Targeting TRPV1 signaling: Galangin improves ethanol-induced gastric mucosal injury

Galangin, a bioactive compound extracted from Alpinia officinarum Hance (Zingiberaceae), a plant with significant ethnopharmacological importance, has been used for thousands of years as a spice, condiment, and medicinal agent for various conditions, including gastrointestinal disorders. Although there is evidence suggesting its potential to improve gastric ulcers, the molecular mechanisms underlying its anti-ulcer properties are not fully understood. of the Study: This study aimed to investigate the effects of galangin on ethanol-induced acute gastric mucosal injury (AGMI) in mice and elucidate its molecular mechanisms. Sixty BALB/c mice were randomly assigned into two main groups: a normal control group (n=10) and an ethanol-induced group (n=50). After establishing the AGMI model in mice using a combination of 40% ethanol and anhydrous ethanol, the ethanol-induced group was further subdivided into five subgroups (n=10): an omeprazole control group (20mg/kg), an untreated ethanol group, and three treatment groups receiving high-dose (50mg/kg) or low-dose (25mg/kg) galangin or capsazepine (CPZ, 2mg/kg). The protective effects of galangin were evaluated through mucosal injury indices, hematoxylin and eosin staining, and quantification of inflammatory markers (IL-1β, IL-6, IL-8, and TNF-α). Oxidative stress levels and matrix metalloproteinase activity were measured using specific assay kits. Molecular docking was conducted to assess the binding affinity of galangin to key proteins within the transient receptor potential vanilloid 1 (TRPV1) pathway. Real-time fluorescence quantitative PCR (qPCR) was used to determine mRNA expression levels of TRPV1, calmodulin (CaM), substance P (SP), and CGRP in gastric tissues. Protein expression levels of TRPV1, nerve growth factor (NGF), tropomyosin receptor kinase A (TRKA), transforming growth factor beta (TGF-β), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) were assessed through Western blot analysis. In cellular experiments, Culture of Human Gastric Epithelial Cells (GES-1) were treated with various concentrations of galangin after 7% ethanol induction. Cell proliferation, apoptosis, and migration were evaluated using Hoechst 33258 staining and transwell migration assays. TRPV1 protein expression was detected using immunofluorescence, and the expression levels of Bcl-2, BCL2-Associated X (BAX), and Caspase-3 were quantified by qPCR. Additionally, specific probe kits were used to measure intracellular calcium ions (Ca2+) and mitochondrial membrane potential. The findings indicate that galangin significantly improved mucosal pathology by reducing ulcer indices and inflammatory levels, while enhancing superoxide dismutase (SOD) activity and decreasing malondialdehyde (MDA) concentration. Galangin also reduced matrix metalloproteinase-2 (MMP-2), m metalloproteinase-9 (MMP-9) levels, promoting mucosal repair. At the cellular level, galangin decreased intracellular calcium ion concentration and mitigated the decline in mitochondrial membrane potential, enhance the restoration of mucosal cells, increased migration and proliferation, and reduced apoptosis. Molecularly, galangin demonstrated favorable binding to TRPV1, NGF, TRKA, TGF-β, COX-2, and NF-κB, and reversed the elevated expression of these proteins. Additionally, galangin downregulated the mRNA expression of TRPV1, CaM, SP, CGRP, BAX, and Caspase-3 in gastric tissues/cells, while upregulating Bcl-2 mRNA expression. Galangin mitigates AGMI by inhibiting the overactivation of the TRPV1 pathway, thereby blocking aberrant signal transduction. This study suggests that galangin has therapeutic potential against ethanol-induced AGMI and may be a viable alternative for the treatment of alcohol-induced gastric mucosal injuries.

Bone Marrow Mesenchymal Stem Cells-derived Exosomes Promote Survival of Random Flaps in Rats through Nrf2-mediated Antioxidative Stress.

Random flaps are the most used defect repair method for head and neck tumors and trauma plastic surgery. The distal part of the flap often undergoes oxidative stress (OS), ultimately leading to flap necrosis. Stem cells' exosomes exhibit potential effects related to anti-inflammatory, regenerative, and antioxidant properties. Nuclear factor erythroid-2-related factor 2 (Nrf2) is an important factor in regulating oxidative balance. Exosomes have been reported to monitor its transcription to alleviate OS. This study examined the impacts and underlying mechanisms of antioxidant actions of exosomes derived from bone marrow mesenchymal stem cells (BMSCs-Exo) on random flaps. BMSCs-Exo were injected into the tail veins of rats on days 0, 1, and 2 after surgery of random flaps. The rats were euthanized on day 3 to calculate the survival rate. Immunohistochemical staining, western blotting, dihydroethidium probe, superoxide dismutase, and malondialdehyde assay kits were used to detect the OS level. Human umbilical vein endothelial cells were cocultured with BMSCs-Exo and ML385 (an inhibitor of Nrf2) in vitro. BMSCs-Exo may significantly improve the survival rate of the random flaps by reducing apoptosis, inflammation, and OS while increasing angiogenesis. Besides, BMSCs-Exo can also increase mitochondrial membrane potential and reduce reactive oxygen species levels in vitro. These therapeutic effects might stem from the activation of the Kelch-like enyol-CoA hydratase (ECH)-associated protein 1 (Keap1)/Nrf2 signaling pathway. BMSCs-Exo improved the tissue antioxidant capacity by regulating the Keap1/Nrf2 signaling pathway. BMSCs-Exo may be a new strategy to solve the problem of random flap necrosis.

Salidroside Inhibits α-Amanitin-Induced AML-12 Cell Apoptosis via the Regulation of PINK1/Parkin-Mediated Mitophagy and Mitochondrial Function.

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.

Targeting the Ferroptosis and Endoplasmic Reticulum Stress Signaling Pathways by CBX7 in Myocardial Ischemia/reperfusion Injury.

Ferroptosis and endoplasmic reticulum stress (ERS) are common events in the process of myocardial ischemia/reperfusion injury (IRI). The suppression of chromobox7 (CBX7) has been reported to protect against ischemia/reperfusion injury, This research is purposed to expose the impacts and mechanism of CBX7 in myocardial IRI. CBX7 expression was detected using RT-qPCR and western blotting analysis. CCK-8 assay detected cell viability. Inflammatory response and oxidative stress were detected by ELISA, DCFH-DA probe and related assay kits. Flow cytometry analysis and caspase3 activity assay were used to detect cell apoptosis. C11-BODIPY 581/591 staining and ferro-orange staining were used to detect lipid reactive oxygen species (ROS) and Fe2+ level, respectively. Western blotting was used to detect the expression of proteins associated with apoptosis, ferroptosis and ERS. In the hypoxia/reoxygenation (H/R) model of rat cardiomyocytes H9c2, CBX7 was highly expressed. CBX7 interference significantly protected against inflammatory response, oxidative stress, apoptosis, ferroptosis and ERS induced by H/R in H9c2 cells. Moreover, after the pretreatment with ferroptosis activator erastin or ERS agonist Tunicamycin (TM), the protective effects of CBX7 knockdown on the inflammation, oxidative stress and apoptosis in H/R-induced H9c2 cells was partially abolished. To summarize, CBX7 down-regulation may exert anti-ferroptosis and anti-ERS activities to alleviate H/R-stimulated myocardial injury.

Abstract A007: Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across >50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Detection of EWSR1 gene rearrangement by fluorescence in situ hybridization in bone and soft tissue tumors: clinical application evaluation and atypical signal analysis

Objective: To investigate the clinical application of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) in bone and soft tissue tumors and to analyze the cases with atypical signal pattern. Methods: The cases detected for EWSR1 gene rearrangement by FISH in Beijing Jishuitan Hospital, Capital Medical University from 2014 to 2021 were collected, and the value of detecting EWSR1 gene rearrangement for diagnosing bone and soft tissue tumors was analyzed. The cases with atypical positive signals were further analyzed by next generation sequencing (NGS). Results: FISH using EWSR1 break-apart probe kit was successfully performed in 97% (205/211) of cases, 6 cases failed. Four of the 6 failures were due to improper decalcification, 1 case due to signal overlap caused by thick slices, and 1 case due to signal amplification and disorder. EWSR1 gene rearrangements were positive in 122 cases (122/205, 59%), atypical positive signal in 8 cases (8/205, 4%), and negative in 75 cases (75/205, 37%). In cases testing positive, the percentage of positive cells ranged from 34% to 98%, with 120 cases (120/122, 98%) showing a positive cell percentage greater than 50%. Among the 205 successfully tested cases, 156 cases were histologically diagnosed as Ewing's sarcoma, of which 110 were positive (110/156, 71%), 7 were atypical positive (7/156, 4%), and 39 were negative (39/156, 25%). Nine cases were histologically diagnosed as clear cell sarcoma of soft tissue, of which 6 were positive (6/9), 1 was atypical positive (1/9), and 2 were negative (2/9). Five cases were histologically diagnosed as extraskeletal myxoid chondrosarcoma, of which 2 were positive (2/5) and 3 were negative (3/5). Three cases were histologically diagnosed as angiomatoid fibrous histiocytoma, of which 2 were positive (2/3) and 1 was negative (1/3). Two cases were histologically diagnosed as myoepithelioma of soft tissue, of which 1 was positive (1/2) and 1 was negative (1/2). One case was histologically diagnosed as olfactory neuroblastoma with a positive result. The 29 other tumor cases including osteosarcoma, synovial sarcoma, and malignant melanoma and others were all negative. Basing on histology as the standard for diagnosis and considering atypical positive cases as negative, comparing with the 29 cases of other tumors as control group, the sensitivity for diagnosing Ewing's sarcoma through the detection of EWSR1 gene rearrangement was 71%, and the specificity was 100%; the sensitivity for diagnosing clear cell sarcoma of soft tissue was 67%, and the specificity was 100%; the sensitivity for diagnosing extraskeletal myxoid chondrosarcoma was 40%, and the specificity was 100%; the sensitivity for diagnosing angiomatoid fibrous histiocytoma was 67%, and the specificity was 100%; the sensitivity for diagnosing myoepithelioma of soft tissue was 50%, and the specificity was 100%; the sensitivity for diagnosing olfactory neuroblastoma was 100%, and the specificity was 100%. Four of 8 cases with atypical positive signals analyzed by NGS showed EWSR1 rearrangement, including EWSR1::FLI1 in one case of Ewing sarcoma, EWSR1::NFATC2 in one case of EWSR1::NFATC2-rearranged sarcoma, EWSR1::ATF1 in one case of clear cell sarcoma of soft tissue and EWSR1::NR4A3 in one case of extraskeletal myxoid chondrosarcoma. Conclusions: Detection of EWSR1 rearrangement by FISH is of utmost significance in the diagnosis of bone and soft tissue tumors. Cases with atypical positive signals should be further scrutinized, correlating with their histomorphology and verifying by NGS if necessary.

Baicalein Alleviates Arsenic-induced Oxidative Stress through Activation of the Keap1/Nrf2 Signalling Pathway in Normal Human Liver Cells.

Oxidative stress is a key mechanism underlying arsenicinduced liver injury, the Kelch-like epichlorohydrin-related protein 1 (Keap1)/nuclear factor E2 related factor 2 (Nrf2) pathway is the main regulatory pathway involved in antioxidant protein and phase II detoxification enzyme expression. The aim of the present study was to investigate the role and mechanism of baicalein in the alleviation of arsenic-induced oxidative stress in normal human liver cells. Normal human liver cells (MIHA cells) were treated with NaAsO2 (0, 5, 10, 20 μM) to observe the effect of different doses of NaAsO2 on MIHA cells. In addition, the cells were treated with DMSO (0.1%), NaAsO2 (20 μM), or a combination of NaAsO2 (20 μM) and Baicalein (25, 50 or 100 μM) for 24 h to observe the antagonistic effect of Baicalein on NaAsO2. Cell viability was determined using a Cell Counting Kit- 8 (CCK-8 kit). The intervention doses of baicalein in subsequent experiments were determined to be 25, 50 and 100μM. The intracellular content of reactive oxygen species (ROS) was assessed using a 2',7'-dichlorodihydrofluorescein diacetate (DCFHDA) probe kit. The malonaldehyde (MDA), Cu-Zn superoxide dismutase (Cu-Zn SOD) and glutathione peroxidase (GSH-Px) activities were determined by a test kit. The expression levels of key genes and proteins were determined by real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blotting. Baicalein upregulated the protein expression levels of phosphorylated Nrf2 (p-Nrf2) and nuclear Nrf2, inhibited the downregulation of Nrf2 target genes induced by arsenic, and decreased the production of ROS and MDA. These results demonstrate that baicalein promotes Nrf2 nuclear translocation by upregulating p-Nrf2 and inhibiting the downregulation of Nrf2 target genes in arsenic-treated MIHA cells, thereby enhancing the antioxidant capacity of cells and reducing oxidative stress. Baicalein alleviated arsenic-induced oxidative stress through activation of the Keap1/Nrf2 signalling pathway in normal human liver cells.

Overexpression of LncRNA MEG3 promotes ferroptosis and enhances chemotherapy sensitivity of hepatocellular carcinoma cells to cisplatin

To investigate the effect of overexpression of LncRNA MEG3 on proliferation, migration and cisplatin sensitivity of hepatoma cells HepG2 and LM3 and explore the underlying and mechanism. The expression of MEG3 in healthy individuals and patients with hepatocellular carcinoma (HCC) was analyzed by online bioinformatics analysis, and Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect MEG3 expression in different HCC cell lines. A MEG3-overexpresing plasmid was transfected in HepG2 and LM3 cells, and the changes in cell proliferation and migration were examined using CCK8 assay and scratch assay. CCK8 assay was used to determine the inhibitory rate of cisplatin on the transfected cells. A reactive oxygen species (ROS) fluorescence probe (DCFH-DA) and malondialdehyde (MDA) kit were used to assess the changes in ROS production and MDA level in the cells. Western blotting was performed to detect the expression levels of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1). The expression of MEG3 was significantly lower in HCC cells than in LO2 cells, which was consistent with the results of bioinformatic analysis (P < 0.05). Overexpression of MEG3 in the HCC cell lines significantly suppressed cell proliferation and migration (P < 0.05), increased the cell inhibition rate of cisplatin (P < 0.05), enhanced cellular ROS production and increased MDA levels in the cells (P < 0.05). MEG3 overexpression significantly decreased the expressions of GPX4 and FTH1 in the HCC cell lines. The expression of MEG3 is decreased in HCC cells, and its overexpression inhibits proliferation and migration and enhances cisplatin sensitivity of HCC cells by promoting ferroptosis of the cells.

Circ-CARD6 inhibits oxidative stress-induced apoptosis and autophagy in ARPE-19 cells via the miR-29b-3p/PRDX6/PI3K/Akt axis

BackgroundOxidative stress-induced damage and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD) and hereditary retinopathy diseases (HRDs). This study aimed to elucidate the roles and mechanisms of circ-CARD6 and miR-29b-3p in oxidative stress-induced RPE and provide new ideas for the diagnosis and treatment of retinopathy disease (RD). MethodsA model of oxidative stress-induced RPE (ARPE-19) was established, and the level of malondialdehyde (MDA) and concentration of reactive oxygen species (ROS) were detected by a DCFH-DA fluorescent probe and MDA kit. The cell viability was measured by a CCK-8 assay. The expression of PRDX6/PI3K/Akt axis genes and proteins related to apoptosis and autophagy were determined by RT‒qPCR and Western blot analyses. The dual-luciferase reporter system confirmed the targeting relationship between miR-29b-3p and circ-CARD6 and between miR-29b-3p and PRDX6. ResultsIn H2O2-treated ARPE-19 cells, the expression of circ-CARD6 and PRDX6 was decreased, while the expression of miR-29b-3p was increased. The overexpression of circ-CARD6 inhibits oxidative stress-induced increases in ROS, apoptosis and autophagy in ARPE-19 cells. circ-CARD6 targets miR-29b-3p, miR-29b-3p targets PRDX6, and circ-CARD6 regulates PRDX6 via miR-29b-3p. Further studies showed that circ-CARD6 acts as a competitive endogenous RNA of miR-29b-3p to affect the expression of PRDX6, thereby inhibiting autophagy and apoptosis in ARPE-19 cells. Conclusioncirc-CARD6 can inhibit oxidative stress and apoptosis by regulating the miR-29b-3p/PRDX6/PI3K/Akt axis.

LiaSR two-component system modulates the oxidative stress response in Streptococcusmutans

Many commensal bacteria of the human oral microbiome can produce reactive oxygen species (ROS). ROS will inhibit the colonization of Streptococcusmutans (S.mutans), a major pathogenic bacteria in dental caries. The LiaSR two-component system in S.mutans can sense and respond to environmental oxidative stress. However, the molecular details of the LiaSR two-component system and oxidative stress response have been unclear. In this study, we aimed to elucidate the underlying mechanisms of the LiaSR two-component system and the mediated oxidative stress response in S.mutans. We performed the H2O2 killing assay, Confocal laser scanning microscopy, and 2,7-Dichlorofluoresce diacetate staining assay to evaluate the sensitivity of S.mutans to H2O2. The propidium iodide probe and TUNEL kit were used to detect the membrane permeability and DNA fragmentation. Quantitative real-time PCR was conducted to analyze the expression level of underlying regulated genes. The liaS and liaR deficient mutants were particularly sensitive to H2O2 compared to their wild strain S.mutans 593, which was previously isolated from a caries-active patient. The intracellular levels of ROS and membrane permeability increased in the mutants. The TUNEL assay showed that the rate of DNA fragmentation in the liaR mutant was higher compared to the wild strain and liaS mutant. Relative expression of the spxA2 gene in the mutants was lower than in the wild strain. The dpr and dinB genes were downregulated in the liaR mutant. These results indicated that the LiaSR two-component system mediated influence on spxA2 expression in S.mutans and contributed to membrane homeostasis, which was involved in the oxidative response process. S.mutans could also elevate the dpr and the dinB genes, which depend on the liaR component in the LiaSR system, may help reduce the DNA damage caused by ROS. This study provides valuable insights into the mechanisms of the LiaSR two-component system in the oxidative stress response of S.mutans.

Perfluorooctane sulfonate (PFOS), a novel environmental pollutant, induces liver injury in mice by activating hepatocyte ferroptosis

ObjectiveWe investigated the toxicological mechanism by which perfluorooctane sulfonate (PFOS) induces liver injury via ferroptosis. MethodsPrimary mouse hepatocytes were treated with LD50 = 55 M PFOS. Their cytotoxicity was detected by CCK-8 and LDH assays, while JC-1 staining was used to identify their mitochondrial membrane potential. GSH-Px, MDA, and SOD levels were determined using kits, and Fe2 + levels were determined using the FerroOrange probe and iron ion assay kit. The DCFH-DA probe was used to examine ROS levels, while Western blot was used to examine protein expressions. After treatment with the ferroptosis inhibitor YL939 and the ROS inhibitor BABTA, the ability of PFOS to induce ferroptosis was observed. In animal experiments, we examined the liver function and the degree of hepatic ferroptosis of mice treated with PFOS as well as their histopathological changes. ResultsPFOS could induce hepatocyte ferroptosis, while YL939 and BABTA treatment could inhibit PFOS-induced ferroptosis. PFOS could induce liver injury and increase ferroptosis levels in tissue, while treatment with YL939 and BABTA could inhibit liver injury. ConclusionPFOS can induce ferroptosis in hepatocytes and cause liver injury in mice, which is its toxicological mechanism.

Theoretical modeling on the temperature-dependent dielectric properties of concrete pavement materials

The variations of concrete pavement temperature will affect the dielectric properties of concrete, which have a substantial influence on the wireless power transfer efficiency of concrete electrified road and non-destructive accuracy of ground-penetrating radar (GPR). In order to elucidate and describe the variations in the dielectric properties of concrete with temperature, the dielectric properties of aggregates (limestone), six types of cement mortar (matrix) and concrete (two types of Portland Cement: P.O 32.5 and P.O 42.5; three W/C ratios: 0.4, 0.5 and 0.6) within the temperature of −30 ℃ to 60 ℃ were acquired via a network analyzer platform and an open-ended coaxial probe kit. The results show that the dielectric constants of limestone, cement mortar and concrete increase with varying degrees when temperature rises. Furthermore, strong linear agreement between the dielectric constants of limestone, cement mortar and concrete and temperature is identified, indicating that the variations in the dielectric properties of concrete with temperature should be taken into consideration. Meanwhile, a temperature-dependent dielectric model of concrete is established by introducing the dielectric-temperature coefficients so as to quantify the influence of temperature on the dielectric properties of concrete. Compared with the traditional models (Brown model, CRIM model, Looyenga model, and Rayleigh model), the proposed model has the highest precision and is less sensitive to temperature variations. Therefore, the proposed model is more suitable for predicting the dielectric properties of concrete at different temperatures, which is essential for the development of future electrified road and promoting non-destructive accuracy of GPR equipment.

Clinicopathological and prognostic features of subungual melanoma in situ

Objective: To investigate the clinicopathological characteristics, immunohistochemical profiles, molecular features, and prognosis of subungual melanoma in situ (SMIS). Methods: Thirty cases of SMIS were collected in Fudan University Shanghai Cancer Center, Shanghai, China from 2018 to 2022. The clinicopathological characteristics and follow-up data were retrospectively analyzed. Histopathologic evaluation and immunohistochemical studies were carried out. By using Vysis melanoma fluorescence in situ hybridization (FISH) probe kit, combined with 9p21(CDKN2A) and 8q24(MYC) assays were performed. Results: There were 8 males and 22 females. The patients' ages ranged from 22 to 65 years (median 48 years). All patients presented with longitudinal melanonychia involving a single digit. Thumb was the most commonly affected digit (16/30, 53.3%). 56.7% (17/30) of the cases presented with Hutchinson's sign. Microscopically, melanocytes proliferated along the dermo-epithelial junction. Hyperchromatism and nuclear pleomorphism were two of the most common histological features. The melanocyte count ranged from 30 to 185. Most cases showed small to medium nuclear enlargement (29/30, 96.7%). Pagetoid spread was seen in all cases. Intra-epithelial mitoses were identified in 56.7% (17/30) of the cases. Involvement of nailfold was found in 19 cases, 4 of which were accompanied by cutaneous adnexal extension. The positive rates of SOX10, PNL2, Melan A, HMB45, S-100, and PRAME were 100.0%, 100.0%, 96.0%, 95.0%, 76.9%, and 83.3%, respectively. FISH analysis was positive in 6/9 of the cases. Follow-up data were available in 28 patients, and all of them were alive without disease. Conclusions: SMIS mainly shows small to medium-sized cells. High melanocyte count, hyperchromatism, nuclear pleomorphism, Pagetoid spreading, intra-epithelial mitosis, nailfold involvement, and cutaneous adnexal extension are important diagnostic hallmarks. Immunohistochemistry including SOX10 and PRAME, combined with FISH analysis, is valuable for the diagnosis of SMIS.

Effect of SLC7A11 gene downregulation on the gefitinib resistance of lung adenocarcinoma PC9/GR cells and its mechanism

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.

Pogostone alleviates angiotensin II-induced cardiomyocyte hypertrophy in H9c2 cells through MAPK and Nrf2 pathway

Purpose: To investigate the effect of pogostone on cardiac hypertrophy.Methods: An in vitro model of myocardial hypertrophy was first established by stimulating H9c2 (rat cardiomyocytes) with angiotensin II (Ang II), and the cells treated with or without pogostone. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured by western blot. Immunofluorescence staining was performed for α-actinin while cell surface area was quantified. Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe and malondialdehyde (MDA) assay kit were used to determine reactive oxygen species (ROS) and MDA levels respectively. Apoptosis was evaluated by flow cytometry while Nrf2, p38, ERK, and JNK protein expression levels were determined by western-blot assay.Results: Compared with the control group, ANP and BNP protein expression levels, cell surface area, ROS, MDA, and apoptosis were all elevated in H9c2 cells after stimulation with Ang II (p &lt; 0.001). Varying doses of pogostone decreased protein expressions of ANP and BNP, reduced cell surface area, decreased ROS and MDA levels, and inhibited apoptosis. Pogostone also up-regulated and inhibited the phosphorylation levels of p38 and ERK, and JNK levels in H9c2 cells.Conclusion: Pogostone reduces protein expression of ANP and BNP and up-regulated Nrf2 protein expression in H9c2 cells stimulated with angiotensin II.