Staining Analysis Research Articles

Phylogenetic trends in TZ staining analysis of six deep dormancy seeds

Abstract The assessment of seed quality and physiological potential is essential in seed production and crop breeding. In the process of rapid detection of seed viability using tetrazolium (TZ) staining, it is necessary to spend a lot of labour and material resources to explore the pretreatment and staining methods of hard and solid seeds with physical barriers. This study explores the TZ staining methods of six hard seeds (Tilia miqueliana, Tilia henryana, Sassafras tzumu, Prunus subhirtella, Prunus sibirica, and Juglans mandshurica) and summarizes the TZ staining conditions required for hard seeds by combining the difference in fat content between seeds and the kinship between species, thus providing a rapid viability test method for the protection of germplasm resources of endangered plants and the optimization of seed bank construction. The TZ staining of six species of hard seeds requires a staining temperature above 35 °C and a TZ solution concentration higher than 1%. Endospermic seeds require shorter staining times than exalbuminous seeds. The higher the fat content of the seeds, the lower the required incubation temperature and TZ concentration for staining, and the longer the staining time. And the closer the relationship between the two species, the more similar their staining conditions become. The TZ staining method of similar species can be predicted according to the genetic distance between the phylogenetic trees, and the viability of new species can be detected quickly.

TXNIP knockdown protects rats against bupivacaine-induced spinal neurotoxicity via the inhibition of oxidative stress and apoptosis

Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

The expression of PKM1 and PKM2 in developing, benign, and cancerous prostatic tissues

BackgroundNeuroendocrine prostate cancer (NEPCa) is the most aggressive type of prostate cancer (PCa). However, energy metabolism, one of the hallmarks of cancer, in NEPCa has not been well studied. Pyruvate kinase M (PKM), which catalyzes the final step of glycolysis, has two main splicing isoforms, PKM1 and PKM2. The expression pattern of PKM1 and PKM2 in NEPCa remains unknown.MethodsIn this study, we used immunohistochemistry, immunofluorescence staining, and bioinformatics analysis to examine the expression of PKM1 and PKM2 in mouse and human prostatic tissues.ResultsWe found that PKM2 was the predominant isoform expressed throughout prostate development and PCa progression, with slightly reduced expression in murine NEPCa. PKM1 was mostly expressed in stromal cells but low-level PKM1 was also detected in prostate basal epithelial cells. Its expression was absent in the majority of prostate adenocarcinoma (AdPCa) specimens but present in a subset of NEPCa. Additionally, we evaluated the mRNA levels of ten PKM isoforms that express exon 9 (PKM1-like) or exon 10 (PKM2-like). Some of these isoforms showed notable expression levels in PCa cell lines and human PCa specimens.DiscussionOur study characterized the expression pattern of PKM1 and PKM2 in prostatic tissues including developing, benign, and cancerous prostate. These findings lay the groundwork for understanding the metabolic changes in different PCa subtypes.

Loss of ZNF451 mediates fibroblast activation and promotes lung fibrosis

BackgroundNo effective therapies for pulmonary fibrosis (PF) exist because of the unclear molecular pathogenesis and the lack of effective therapeutic targets. Zinc finger protein 451 (ZNF451), a transcriptional regulator, plays crucial roles in the pathogenesis of several diseases. However, its expression pattern and function in PF remain unknown. This study was designed to investigate the role of ZNF451 in the pathogenesis of lung fibrosis.MethodsGEO dataset analysis, RT‒PCR, and immunoblot assays were used to examine the expression of ZNF451 in PF; ZNF451 knockout mice and ZNF451-overexpressing lentivirus were used to determine the importance of ZNF451 in PF progression; and migration assays, immunofluorescence staining, and RNA-seq analysis were used for mechanistic studies.ResultsZNF451 is downregulated and negatively associated with disease severity in PF. Compared with wild-type (WT) mice, ZNF451 knockout mice exhibited much more serious PF changes. However, ZNF451 overexpression protects mice from BLM-induced pulmonary fibrosis. Mechanistically, ZNF451 downregulation triggers fibroblast activation by increasing the expression of PDGFB and subsequently activating PI3K/Akt signaling.ConclusionThese findings uncover a critical role of ZNF451 in PF progression and introduce a novel regulatory mechanism of ZNF451 in fibroblast activation. Our study suggests that ZNF451 serves as a potential therapeutic target for PF and that strategies aimed at increasing ZNF451 expression may be promising therapeutic approaches for PF.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 μM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell–cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

Abstract LB434: Rapid and reproducible expansion of rare tumor cells: The R3CE platform for personalized medicine

Abstract We have developed a novel single-cell derived 3D organoid culture platform, Rapid, Reproducible, Rare Cell 3D Expansion (R3CE), capable of generating 3D organoids from solid tumors and normal tissues obtained via surgical tissues and needle biopsies within one week. This method demonstrates over 90% success in banking for at least two passages with over 106 cells across various cancer types, including breast cancer, colorectal cancer, hepatoma cancer, and ovarian cancer. The organoids maintain high fidelity to original tissues, as evidenced by H&E staining, WES, and protein marker analysis across generations. Notably, the R3CE platform can also cultivate circulating tumor cells (CTCs) from peripheral blood. The drug sensitivity profiles of RCE-cultured CTCs closely mirrored clinical outcomes in breast cancer patients. For a patient with stage IV HER2-positive breast cancer, our real-time assay results led to an adjustment in treatment from lapatinib/5-FU to lapatinib/Trastuzumab emtansine (TDM-1), which resulted in substantial clinical improvement. To our knowledge, the R3CE platform is the first 3D organoid system that does not require aggregation or 3D scaffold such as matrigel system. These advancements position the R3CE platform as a promising tool for personalized medicine, offering a rapid and accurate method for drug screening and therapeutic response prediction to guide clinical decision-making in cancer treatment. Citation Format: Wen-Hung Kuo, Yen-Jang Huang, Jia-Yang Chen, Yi-Chun Wu, Chia-Chun Chen, Mark D. Pegram, Ying-Chih Chang. Rapid and reproducible expansion of rare tumor cells: The R3CE platform for personalized medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB434.

Effects of fluorene-9-bisphenol exposure on anxiety-like and social behavior in mice and protective potential of exogenous melatonin.

Fluorene-9-bisphenol (BHPF) is widely used in the manufacture of plastic products and potentially disrupts several physiological processes, but its biological effects on social behavior remain unknown. In this study, we investigated the effects of BHPF exposure on anxiety-like and social behavior in female mice and the potential mechanisms, thereby proposing a potential therapy strategy. We exposed female Balb/c mice to BHPF by oral gavage at different doses (0.5, 50 mg/kg bw/2-day) for 28 days, which were found BHPF (50 mg/kg) exposure affected motor activity in the open field test (OFT) and elevated cross maze (EPM), resulting in anxiety-like behaviors, as well as abnormal social behavioral deficits in the Social Interaction Test (SIT). Analysis of histopathological staining results showed that BHPF exposure caused damage to hippocampal neurons in the CA1/CA3/DG region and decreased Nissl pyramidal neurons in the CA1/CA3 regions of the hippocampus, as well as a decrease in parvalbumin neuron expression. In addition, BHPF exposure upregulated the expression of excitatory and inhibitory (E/I) vesicle transporter genes (Vglut1, Vglut2, VGAT, GAD67, Gabra) and axon growth gene (Dcc) in the mouse hippocampus. Interestingly, behavioral disturbances and E/I balance could be alleviated by exogenous melatonin (15 mg/kg bw/2-day) therapy. Our findings suggest that exogenous melatonin may be a potential therapy with protective potential for ameliorating or preventing BHPF-induced hippocampal neuronal damage and behavioral disturbances. This study provided new insight into the neurotoxicological effects on organisms exposed to endocrine-disrupting chemicals and aroused our vigilance in current environmental safety about chemical use.

Scutellarein Ameliorated Chondrocyte Inflammation and Osteoarthritis in Rats.

Osteoarthritis (OA) is a degenerative joint disorder characterized by the gradual degradation of joint cartilage and local inflammation. This study aimed to investigate the anti-OA effect of scutellarein (SCU), a single-unit flavonoid compound obtained from Scutellaria barbata D. Don, in rats. The extracted rat chondrocytes were treated with SCU and IL-1β. The chondrocytes were divided into control group, IL-1β group, IL-1β+SCU 50 µmol/L group, and IL-1β+SCU 100 µmol/L group. Morphology of rat chondrocytes was observed by toluidine blue and safranin O staining. CCK-8 method was used to detect the cytotoxicity of SCU. ELISA, qRT-PCR, Western blotting, immunofluorescence, SAβ-gal staining, flow cytometry, and bioinformatics analysis were applied to evaluate the effect of SCU on rat chondrocytes under IL-1β intervention. Additionally, anterior cruciate ligament transection (ACL-T) was used to establish a rat OA model. Histological changes were detected by safranin O/fast green, hematoxylin-eosin (HE) staining, and immunohistochemistry. SCU protected cartilage and exhibited anti-inflammatory effects via multiple mechanisms. Specifically, it could enhance the synthesis of extracellular matrix in cartilage cells and inhibit its degradation. In addition, SCU partially inhibited the nuclear factor kappa-B/mitogen-activated protein kinase (NF-κB/MAPK) pathway, thereby reducing inflammatory cytokine production in the joint cartilage. Furthermore, SCU significantly reduced IL-1β-induced apoptosis and senescence in rat chondrocytes, further highlighting its potential role in OA treatment. In vivo experiments revealed that SCU (at a dose of 50 mg/kg) administered for 2 months could significantly delay the progression of cartilage damage, which was reflected in a lower Osteoarthritis Research Society International (OARSI) score, and reduced expression of matrix metalloproteinase 13 (MMP13) in cartilage. SCU is effective in the therapeutic management of OA and could serve as a potential candidate for future clinical drug therapy for OA.

Modulation of the Cellular Microenvironment by Mechanical Fluid Shear Stress and Hypoxia Alters the Differentiation Capacity of Skeletal Muscle-Derived Stem Cells

Skeletal muscle-derived stem cells (MDSCs) are the key modulators of muscle regeneration. An inappropriate cellular microenvironment can reduce the regenerative capacity of MDSCs. This study evaluates the effect of microenvironmental alterations on the cell differentiation capacity using either mechanical fluid shear stress (FSS) or hypoxic conditions. C2C12 mouse myoblasts were differentiated under cyclic FSS (CFSS), periodic FSS (PFSS) for one hour, and hypoxia (3% O2) for up to seven days. Cell proliferation and myogenic differentiation capacities were evaluated using cell viability assays, immunohistochemical staining, and morphometric analysis. The expression of MyoD, myogenin, myosin heavy chain, nitric oxide, hypoxia-inducible factor 1 alpha (HIF1α), vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) was quantified by means of RT-qPCR. The data showed that FSS conditions altered cell morphology and increased cell viability and cell distribution compared to static conditions. MyoD and myogenin expression was upregulated under both FSS conditions. CFSS induction improved myogenic differentiation parameters including myotube number, size and fusion capacity. Although hypoxia enhanced cell viability compared to normoxia, it reduced differentiation capacity, as indicated by the downregulation of myogenin and mTOR expression, as well as reducing myotube formation. Under hypoxic conditions, increased nitric oxide production and upregulation of VEGF expression were detected for up to 72 h. The data suggest an improved myogenic differentiation capacity under mechanical FSS; in contrast, the cell differentiation capacity was impaired under hypoxic conditions. The data point out that optimizing the biomechanical and oxidative stressors in the cellular microenvironment could improve stem cell transplantation and enhance their regenerative potential in the context of cell-based therapies.

Application of extracellular matrix cross-linked by microbial transglutaminase to promote wound healing

One primary focus of skin tissue engineering has been the creation of innovative biomaterials to facilitate rapid wound healing. Extracellular matrix (ECM), an essential biofunctional substance, has recently been discovered to play a crucial role in wound healing. Consequently, we endeavored to decellularize ECM from pig achilles tendon and refine its mechanical and biological properties through modification by utilizing cross-linking agents. Glutaraldehyde (GA), 1-ethyl-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), double aldol starch (DAS), and microbial transglutaminase (MTG) were utilized to produce crosslinked ECM variants (GA-ECM, EDC/NHS-ECM, DAS-ECM, and MTG-ECM). Comprehensive assessments were conducted to evaluate the physical properties, biocompatibility, and wound healing efficacy of each material. The results indicated that MTG-ECM exhibited superior tensile strength, excellent hydrophilicity, minimal cytotoxicity, and the best pro-healing impact among the four modified scaffolds. Staining analysis of tissue sections further revealed that MTG-ECM impeded the transition from type III collagen to type I collagen in the wound area, potentially reducing the development of wound scar. Therefore, MTG-ECM is expected to be a potential pro-skin repair scaffold material to prevent scar formation.

Metformin normalizes mitochondrial function to delay astrocyte senescence in a mouse model of Parkinson’s disease through Mfn2-cGAS signaling

BackgroundSenescent astrocytes play crucial roles in age-associated neurodegenerative diseases, including Parkinson’s disease (PD). Metformin, a drug widely used for treating diabetes, exerts longevity effects and neuroprotective activities. However, its effect on astrocyte senescence in PD remains to be defined.MethodsLong culture-induced replicative senescence model and 1-methyl-4-phenylpyridinium/α-synuclein aggregate-induced premature senescence model, and a mouse model of PD were used to investigate the effect of metformin on astrocyte senescence in vivo and in vitro. Immunofluorescence staining and flow cytometric analyses were performed to evaluate the mitochondrial function. We stereotactically injected AAV carrying GFAP-promoter-cGAS-shRNA to mouse substantia nigra pars compacta regions to specifically reduce astrocytic cGAS expression to clarify the potential molecular mechanism by which metformin inhibited the astrocyte senescence in PD.ResultsWe showed that metformin inhibited the astrocyte senescence in vitro and in PD mice. Mechanistically, metformin normalized mitochondrial function to reduce mitochondrial DNA release through mitofusin 2 (Mfn2), leading to inactivation of cGAS-STING, which delayed astrocyte senescence and prevented neurodegeneration. Mfn2 overexpression in astrocytes reversed the inhibitory role of metformin in cGAS-STING activation and astrocyte senescence. More importantly, metformin ameliorated dopamine neuron injury and behavioral deficits in mice by reducing the accumulation of senescent astrocytes via inhibition of astrocytic cGAS activation. Deletion of astrocytic cGAS abolished the suppressive effects of metformin on astrocyte senescence and neurodegeneration.ConclusionsThis work reveals that metformin delays astrocyte senescence via inhibiting astrocytic Mfn2-cGAS activation and suggest that metformin is a promising therapeutic agent for age-associated neurodegenerative diseases.

[Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection].

To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson's trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson's trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

An in vitro evaluation of Icon resin infiltrant penetration into demineralized enamel lesions using an indirect staining technique with confocal laser scanning microscope analysis in dual fluorescence mode

Abstract Context: White spot lesion is the first clinical sign of a caries lesion and represents mineral loss from the enamel subsurface. Aim: The aim of this study was to evaluate the penetration depth (PD) of Icon resin infiltrant into artificially demineralized enamel lesions using confocal laser scanning microscope (CLSM) analysis in dual fluorescence mode. Settings and Designs: The design of the study was an in vitro study. Materials and Methods: 22 extracted human permanent maxillary central incisor teeth were collected, and enamel sections were obtained from the coronal middle third. All enamel specimens were exposed to demineralization and remineralization solutions for 14 days. On positive confirmation of enamel demineralization by scanning electron microscope analysis, 20 specimens were then subjected to Icon resin infiltration following manufacturer instructions. Specimens were processed with indirect staining technique using rhodamine B and sodium fluorescein dyes and examined under CLSM at ×10 magnification in dual fluorescence mode using ImageJ software to evaluate PD of resin infiltrant into demineralized enamel lesions. Statistical Analysis Used: Obtained data were analyzed using an independent t-test. P ≤0.05 was considered statistically significant. Results: The maximum depth of demineralized enamel lesion was 590 μm, and the mean depth was 290.78 ± 14.80 μm. The maximum depth of resin infiltrant penetration was 580 μm, and the mean depth was 279.08 ± 13.88 μm; P = 0.006. The percentage penetration of resin infiltrant was 95.99%. Conclusion: Icon resin infiltrant was highly effective in its depth of penetration into demineralized enamel lesions. The use of indirect staining and CLSM analysis in dual fluorescence mode is more reliable and accurate technique to evaluate the PD of resin infiltrant.

KIF15 promotes the development and progression of chordoma via activating PI3K-AKT signalling pathway

AimsDespite its implication in various human cancers, the expression and functional significance of Kinesin family member 15 (KIF15) in chordomas remain unexplored. Main methodsThe evaluation of KIF15 protein levels was conducted through immunohistochemistry (IHC) staining and western blot analysis. Cell proliferation was quantified using MTT and CCK8 assays, whereas cell migration was examined using wound healing and Transwell assays. Furthermore, flow cytometric analysis was utilized to assess cell apoptosis and the cell cycle. Additionally, in vivo experiments were performed using a mouse xenograft model. Key findingsOur study revealed significantly higher expression of KIF15 in stage III chordoma tissues compared to stage II tissues. Knockdown of KIF15 led to notable inhibition of cell proliferation and migration, along with enhanced apoptosis and cell cycle arrest. In vivo studies further confirmed the inhibitory effects of KIF15 knockdown on chordoma tumour growth. In terms of mechanism, we identified the involvement of the PI3K-AKT signalling pathway mediated by KIF15 in chordomas. Notably, the anti-tumour effects of KIF15 deficiency on chordomas were partially reversed by the addition of an AKT activator. SignificanceKIF15 promotes chordoma development and progression through the activation of the PI3K-AKT signalling pathway. Thus, targeting KIF15 might be a promising therapeutic strategy for treating chordomas.

Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity

BackgroundRepeated neonatal sevoflurane exposures led to neurocognitive disorders in young mice. We aimed to assess the role of microglia and complement C1q in sevoflurane-induced neurotoxicity and explore the underlying mechanisms.MethodsNeonatal mice were treated with sevoflurane on postnatal days 6, 8, and 10, and the Morris water maze was performed to assess cognitive functions. For mechanistic explorations, mice were treated with minocycline, C1q-antibody ANX005, and sialidase-inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA) before sevoflurane exposures. Western blotting, RT-qPCR, Golgi staining, 3D reconstruction and engulfment analysis, immunofluorescence, and microglial morphology analysis were performed. In vitro experiments were conducted in microglial cell line BV2 cells.ResultsRepeated neonatal sevoflurane exposures resulted in deficiencies in learning and cognition of young mice, accompanied by microglial activation and synapse loss. Sevoflurane enhanced microglia-mediated synapse elimination through C1q binding to synapses. Inhibition of microglial activation and phagocytosis with minocycline significantly reduced the loss of synapses. We further revealed the involvement of neuronal sialic acids in this process. The enhanced activity of sialidase by sevoflurane led to the loss of sialic acids, which facilitated C1q binding to synapses. Inhibition of C1q with ANX005 or inhibition of sialidase with NADNA significantly rescued microglia-mediated synapse loss and improved neurocognitive function. Sevoflurane enhanced the engulfment of BV2 cells, which was reversed by ANX005.ConclusionsOur findings demonstrated that C1q-mediated microglial synaptic elimination by enhancing desialylation contributed to sevoflurane-induced developmental neurotoxicity. Inhibition of C1q or sialidase may be a potential therapeutic strategy for this neurotoxicity.

Molecular characterization of transcription factor CREB3L2 and CREB3L3 and their role in melanogenesis in Pacific oysters (Crassostrea gigas)

Colorful shells in mollusks are commonly attributable to the presence of biological pigments. In Pacific oysters, the inheritance patterns of several shell colors have been investigated, but little is known about the molecular mechanisms of melanogenesis and pigmentation. cAMP-response element binding proteins (CREB) are important transcription factors in the cAMP-mediated melanogenesis pathway. In this study, we characterized two CREB genes (CREB3L2 and CREB3L3) from Pacific oysters. Both of them contained a conserved DNA-binding and dimerization domain (a basic-leucine zipper domain). CREB3L2 and CREB3L3 were expressed highly in the mantle tissues and exhibited higher expression levels in the black-shell oyster than in the white. Masson-Fontana melanin staining and immunofluorescence analysis showed that the location of CREB3L2 protein was generally consistent with the distribution of melanin in oyster edge mantle. Dual-luciferase reporter assays revealed that CREB3L2 and CREB3L3 could activate the microphthalmia-associated transcription factor (MITF) promoter and this process was regulated by the level of cAMP. Additionally, we found that cAMP regulated melanogenic gene expression through the CREB-MITF-TYR axis. These results implied that CREB3L2 and CREB3L3 play important roles in melanin synthesis and pigmentation in Pacific oysters.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor β1/Smad (TGF-β1/Smad) signaling pathway. Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway. A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-β signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).