Cells Stained Research Articles

Anticancer effects of rutin from Fagopyrum tataricum (tartary buckwheat) against osteosarcoma cell line.

The present study is analysisof the seeds of buckwheat (Fagopyrum sp.),member of the Polygonaceae family for isolation of rutin and its anticancer property againstOsteosarcoma celllines (SAOS2). The selected plant is traditionally used for diabetes and cancer. It has several biological properties such as antibacterial, antioxidant and anti-aging. Thirty-five buckwheat cultivars were obtained from Nepal Agriculture Genetic Resources Centre (NAGRC) Khumaltar, Kathmandu, Nepal, andKumrek Sikkim. These plant varieties are scientifically evaluated their biological properties. Rutin wasfractionated from buckwheat seeds using methanol fraction and analysed for quality by HPLC method. The rutin fraction of the cultivar NGRC03731 a tartary buck wheat and standard rutin was used against Osteosarcoma cell lines (SAOS2) and human gingival fibroblast cells (hGFs) for anticancer activity. The cell viability using rutin fraction and standard rutin treated with SAOS2 cells were assessed by MTT assay. For further research, the best doses (IC-50: 20g/ml) were applied. By using AO/EtBr dual staining, the effects of Rutin fraction on SAOS2 cell death were analysed. The scratch wound healing assay was used to analyse cell migration. Real-time PCR was used to analyse the pro-/anti-apoptotic gene expression. The seeds with the highest rutin content, NGRC03731 seeds, had 433mg/100g of rutin.The rutin fraction treatment and standard rutin significantly reduced cell viability in the MTT assay, and osteosarcoma cells were observed on sensitive to the IC-50 dose at a concentration of 20g/ml after 24h.The SAOS2 cells exposed to rutin fraction at 20g/ml and standard rutin at 10g/ml exhibited significant morphological alterations, cell shrinkage and decreased cell density, which indicate apoptotic cells.Rutin-fraction treated cells stained with acridine orange/ethidium bromide (AO/EtBr) dual staining cells turned yellow, orange, and red which indicatesto measure apoptosis.The anti-migration potential of rutin fraction, results prevented the migration of SAOS2 cancer cells.Rutin-fraction significantly increased the expression of pro-apoptotic proteinsBad, using real-time PCR analysis (mRNA for Bcl-2 family proteins) resulted Bcl-2's expression is negatively regulated. Osteosarcoma (SAOS2) cell lines' proliferation, migration, and ability to proliferate were reduced markedly by rutin fraction and it also causes apoptosis of Osteosarcoma cell lines (SAOS2).

The application of a 4D-printed chitosan-based stem cell carrier for the repair of corneal alkali burns

BackgroundCorneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs.MethodsPrepare solutions of chitosan acetate, carboxymethyl chitosan, and β-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections.ResultsUsing the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01).ConclusionIn summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects.Graphical abstract

Novel tubulin polymerization inhibitors based on the hybridization of coumarin and indole ring: Design, synthesis and bioactivities evaluation

In order to find the potent tubulin polymerization inhibitors as anticancer drugs, a series of novel coumarin-indole derivatives were synthesized, and the bioactivity of these synthetic compounds was performed to elucidate the mechanism of anticancer activity. In vitro cell experiments indicated that among these compounds, 7a exhibited excellent inhibitory activities on MCF-7, A549 and HepG2 cells, having IC50 values ranging from 1.79 to 40.55 nanomolar, which was better than the positives cisplatin and colchicine. Tubulin polymerization inhibition assay found 7a could inhibit the polymerization of tubulin. Meanwhile, immunofluorescence staining of A549 cells showed that 7a targeted the tubulin to exert its anticancer activity. Additionally, compound 7a displayed significant inhibition of the wound-healing activity of A549 cells. The outcomes of flow cytometry demonstrated 7a could significantly halt the progression of the cell cycle during the G2/M phase and induce the apoptosis of A549 cells by reducing the mitochondrial membrane potential and increasing the level of ROS. Finally, molecular docking further demonstrated 7a could interact with tubulin.Collectively, the promising biological activity of 7a makes it valuable as further development of antitumor drugs.

Supramolecular dye nanoassemblies for advanced diagnostics and therapies.

Dyes have conventionally been used in medicine for staining cells, tissues, and organelles. Since these compounds are also known as photosensitizers (PSs) which exhibit photoresponsivity upon photon illumination, there is a high desire towards formulating these molecules into nanoparticles (NPs) to achieve improved delivery efficiency and enhanced stability for novel imaging and therapeutic applications. Furthermore, it has been shown that some of the photophysical properties of these molecules can be altered upon NP formation thereby playing a major role in the outcome of their application. In this review, we primarily focus on introducing dye categories, their formulation strategies and how these strategies affect their photophysical properties in the context of photothermal and non-photothermal applications. More specifically, the most recent progress showing the potential of dye supramolecular assemblies in modalities such as photoacoustic and fluorescence imaging, photothermal and photodynamic therapies as well as their employment in photoablation as a novel modality will be outlined. Aside from their photophysical activity, we delve shortly into the emerging application of dyes as drug stabilizing agents where these molecules are used together with aggregator molecules to form stable nanoparticles.

COMPARISON OF HSP90 EXPRESSION IN CONJUNCTIVAL INTRAEPITHELIAL NEOPLASIA AND CONJUNCTIVAL SQUAMOUS CELL CARCINOMA

Abstract&#x0D; Introduction &amp; Objectives : Heat shock protein 90 (HSP90) participates in stabilizing and activating client proteins involved in malignant transformation. This study aims to determine whether there are different values for the expression of HSP90 in Conjunctival Intraepithelial Neoplasia (CIN) and Conjunctival Squamous Cell Carcinoma (CSCC).&#x0D; Methods : A total of 24 OSSN samples, including 15 cases of CIN and 9 cases of CSCC were evaluated. The immunohistochemical expression of HSP90 was assessed using a scoring system based on the percentage of positive staining of nuclear and cytoplasmic cells, then the intensity of staining was evaluated using the German Immunoreactive Score.&#x0D; Results : In the study, the average cytoplasmic Immunoreactive Score in CIN was 9.87 and CSCC was 16.89 with a significant result (p=0.007), while the average nuclear Immunoreactive Score in CIN was 11.30 and CSCC was 14.50 with insignificant results (p=0.274). The results of the correlation test showed that the cytoplasmic expression groups with low categories were all in CIN as much as 6 (100%) so they had a ratio of 6:0. and the high category of HSP90 cytoplasmic expression group was only found in CSCC as many as 2 (100%) samples with a ratio of 0:2. The results of the correlation test using the Pearson test obtained a significant correlation value (p=0.006) with a moderate correlation strength (r=0.545).&#x0D; Conclusion : There are differences in the expression of cytoplasmic and nuclear HSP90 but only the cytoplasmic expression of HSP90 is significantly associated with CIN and CSCC.

Immunohistochemical expression of H3.3 G34W in 100 giant cell tumors of bone and its diagnostic mimics, including its value in resolving uncommon diagnostic scenarios: A single institutional study at a tertiary cancer referral center, India.

There can be a diagnostic challenge in differentiating giant cell tumor of bone (GCTB) from its mimics. Lately, histone H 3 F 3 A (Histone 3.3 ) G34W has been identified as a promising immunohistochemical marker. This study was aimed at evaluating H3.3 G34W immunostaining in 100 GCTBs, including its value in resolving diagnostic dilemmas. Immunohistochemical staining for H3.3 G34W was graded in terms of staining intensity (1+ to 3+) and the percentage of tumor cells showing crisp nuclear staining. One hundred GCTBs occurred in 58 males and 42 females (M: F ratio = 1.3), of 7-66 years age (average = 31.3, median = 28), commonly in distal femur (26), followed by proximal tibia (17), distal radius (12), proximal humerus (7), metacarpals (7), sacrum (6), proximal fibula (6), and relatively unusual sites (19), including a single multicentric case. Out of 92 GCTBs, wherein H3.3 G34W immunostaining worked, 81 (88.1%) showed positive staining in the mononuclear cells, including tumors with fibrous histiocytoma-like areas, sparing osteoclast-like giant cells, with 3+ staining intensity in 65/81 (80%) tumors. All 7/7 (100%) malignant GCTBs showed positive staining, including the pleomorphic/sarcomatous cells. All 7/7 (100%) metastatic GCTBs showed positive immunostaining. Seven out of 10 post-denosumab treated GCTBs showed positive H3.3 G34W immunostaining in the residual mononuclear cells. None of the other 37 "giant cell-rich" lesions displayed H3.3 G34W immunostaining. Four of 9 GCTBs tested for H3.3 G34W mutation showed positive results. The diagnostic sensitivity and specificity of H3.3 G34W for GCTB were 88.1% and 100%, respectively. This constitutes one of the first reports from our country, further validating the diagnostic value of H3.3 G34W in differentiating GCTB, including metastatic and malignant forms from its mimics, including small biopsy samples. Its value in various diagnostic dilemmas is presented and utility in identifying residual tumor cells in post-denosumab treated GCTBs is worth exploring.

EFFECT OF TOPICAL BRIMONIDINE ON NMDA RECEPTOR EXPRESSION AND RETINA GANGLION CELL DENSITY IN TRAUMATIC OPTIC NEUROPATHY RATS MODEL

Abstract&#x0D; Introduction &amp; Objectives : Traumatic optic neuropathy is a visual disturbance caused by acute injury to the optic nerve which results in retinal ganglion cell apoptosis through the glutamate excitotoxicity pathway. Brimonidine is an alpha 2-receptor agonist that acts as a neuroprotector in the glutamate excitotoxicity pathway and causes modulation of NMDA receptors associated with glutamate. Objective of this study is to prove the effect of topical brimonidine on NMDA receptor expression and retinal ganglion cell density in a rat model of traumatic optic neuropathy&#x0D; Methods : Rat models of traumatic optic neuropathy was carried out using the optic nerve crush method. The treatment group was given topical brimonidine 0.15% 1 drop/12 hours for 14 days. NMDA receptor expression was assessed by immunohistochemical staining and retinal ganglion cell density was assessed by Hematoxylin-eosin staining. Statistical analysis was performed to assess the correlation between NMDA receptor expression and retinal ganglion cell density&#x0D; Results : The Allred score of NMDA receptors expression in the treatment group was lower than control group, with significant difference (p=0.002). The mean density of retinal ganglion cells in the treatment and control group was 20,99  7,76 and 12,51  3,10, respectively with a significant difference (p=0.032). There was a significant correlation between NMDA receptor expression and retinal ganglion cell density in the treatment group (p=0.035) with strong negative correlation (r= -0.611)&#x0D; Conclusion : Topical administration of brimonidine to the Wistar rat model of traumatic optic neuropathy can suppress NMDA receptor expression and maintain retinal cell ganglion density

EFFECT OF ORAL CURCUMIN ADMINISTRATION ON NMDA RECEPTOR EXPRESSION AND RETINAL GANGLION CELL DENSITY

Abstract&#x0D; Introduction &amp; Objectives : To prove the effect of oral curcumin on NMDA receptor expression and retinal ganglion cell density.&#x0D; Methods : Wistar rats model of traumatic optic neuropathy were divided into 2 groups. The treatment group was given oral curcumin 150mg/kgBW/24 hours for 14 days 1 hour after trauma. NMDA receptor expression was examined by IHC staining and retinal ganglion cell density was examined by HE staining. Data were collected and analyzed.&#x0D; Results : NMDA expression was found to be significantly lower in the treatment group compared to the control group (p = 0.046). The mean of ganglion density in the control and treatment group was 12.88 ± 2.44 and 17.92 ± 6.95, respectively. The treatment group had a significantly higher ganglion density (p = 0.043). There was a significant relationship between NMDA receptor expression and retinal ganglion density (p = 0.043, r = -0.591).&#x0D; Conclusion : Curcumin can suppress the expression of NMDA receptors to maintain retinal ganglion density in rat models of optic neuropathy

Preparation of Cu2+/TA/HAP composite coating with anti-bacterial and osteogenic potential on 3D-printed porous Ti alloy scaffolds for orthopedic applications.

Because of stress shielding effects, traditional titanium (Ti) alloy scaffolds have a high elastic modulus, which might promote looseness and bone disintegration surrounding the implant, increasing the likelihood of a second surgery. In contrast, 3D-printed porous Ti alloy scaffolds can reduce the scaffold weight while enhancing biocompatibility. Further, these scaffolds' porous nature allows bone tissue ingrowth as well as strong pore connectivity, which can improve nutrient absorption. Nevertheless, bare Ti alloy implants may fail because of inadequate bone integration; hence, adding a coating on the implant surface is an effective technique for improving implant stability. In this study, a composite coating comprising hydroxyapatite (HAP), chitosan (CS), tannic acid (TA) and copper ions (Cu2+) (Cu2+/TA/HAP composite coating) was prepared on the surface of 3D printed porous Ti alloy scaffolds using electrophoretic deposition. Using the standard plate count method, Live/Dead bacteria staining assay, FITC Phalloidin and 4',6-diamidino-2-phenylindole staining assay, and live/dead staining of cells we determined that the composite coating has better antibacterial properties and cytocompatibility as well as lower cytotoxicity. The Alkaline Phosphatase assay revealed that the coating results showed good osteogenesis potential. Overall, the composite coatings produced in this investigation give new potential for the application of Ti alloys in clinics.

Expression of PSMA in Tumor-Associated Vasculature Predicts Poorer Survival in Patients With Hepatocellular Carcinoma and Is Likely Associated With PD-L1.

PSMA (prostate-specific membrane antigen) is a type II transmembrane glycoprotein recently found to be expressed in hepatocellular carcinoma (HCC). We aimed to characterize the expression pattern of PSMA in HCC and its association with clinicopathologic parameters and other biomarkers. Immunohistochemical studies for PSMA were performed on a previously established tissue microarray of 103 surgically resected HCC. Conceivable PSMA expression in ≥5% tumor-associated vasculature (TAV) was considered positive, and was identified in 56 (54.4%) tumors. Eight (7.8%) tumors also showed membranous/cytoplasmic and/or canalicular staining in tumor cells. By chi-square tests, only PSMA-positive TAV was associated with moderate-to-poorly differentiated HCC and the modified higher tumor stage (P < .05). PSMA-positive TAV was not associated with age, sex, or expression of glypican-3, keratin 7, CD3, CD8, HHLA-2, but marginally correlated with programmed death-ligand 1 (PD-L1) expression (P = .052). Kaplan-Meier survival analysis revealed PSMA-positive TAV as an independent risk factor for poorer disease-specific survival (P = .008). Co-expression of PD-L1 did not ameliorate the adverse prognostication of PSMA-positive TAV. Membranous/cytoplasmic/canalicular expression of PSMA alone was not prognostically significant. Our study confirmed that PSMA-positive TAV is a prospective diagnostic and prognostic biomarker for HCC. Co-expression of PSMA with PD-L1 may suggest potential crosstalk between the 2 proteins, likely regulating the tumor microenvironment.

Pericyte activation accompanied by peritubular capillaries dysfunction and pericyte-to-myofibroblast transition is associated with renal fibrosis in diabetic nephropathy.

Tubulointerstitial renal fibrosis is an essential feature of diabetic nephropathy (DN). Pericytes play a critical role in microvascular diseases and renal fibrogenesis. However, the role of pericytes in DN remains unclear. Herein, we aimed to explore the properties and possible mechanisms of pericytes in renal fibrosis in DN. We used multiplex immunofluorescence staining to evaluate the location and expression of activated pericytes and to assess capillary dilation and interstitial fibrosis in the kidneys of db/db mice. Pericytes were co-stained for alpha-smooth muscle actin (α-SMA) to determine which ones differentiate into myofibroblasts in db/db mice. Expression of CD34 and platelet-derived growth factor receptor beta (PDGFR-β) was assessed in kidney tissue from patients with DN by immunohistochemical staining. We found that cell staining for nerve/glial antigen 2 (NG2)+ and PDGFR-β+ was greater in the kidneys of db/db mice than in those of db/m mice. There was impaired pericyte coverage of blood vessels and capillary dilation in the renal interstitium. These changes were accompanied by increased collagen I staining and an increase in the number of pericytes with profibrotic phenotypes, as identified by increased NG2+/PDGFR-β+/α-SMA+ and decreased NG2+/PDGFR-β+/α-SMA- staining. In DN patients, expression of PDGFR-β was stronger and there was loss of CD34 compared with the findings in control patients with minor glomerular lesions. In this study, we demonstrated that pericyte activation accompanied by peritubular capillary dysfunction and pericytemyofibroblast transition is associated with renal fibrosis in DN.

Construction of a g-C3N4/Bi(OH)3 Heterojunction for the Enhancement of Visible Light Photocatalytic Antibacterial Activity.

Photocatalytic technology has been recently conducted to remove microbial contamination due to its unique features of nontoxic by-products, low cost, negligible microbial resistance and broad-spectrum elimination capacity. Herein, a novel two dimensional (2D) g-C3N4/Bi(OH)3 (CNB) heterojunction was fabricated byincorporating Bi(OH)3 (BOH) nanoparticles with g-C3N4 (CN) nanosheets. This CNB heterojunction exhibited high photocatalytic antibacterial efficiency (99.3%) against Escherichia coli (E. coli) under visible light irradiation, which was 4.3 and 3.4 times that of BOH (23.0%) and CN (28.0%), respectively. The increase in specific surface area, ultra-thin layered structure, construction of a heterojunction and enhancement of visible light absorption were conducive to facilitating the separation and transfer of photoinduced charge carriers. Live/dead cell staining, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assays and scanning electron microscopy (SEM) have been implemented to investigate the damage to the cell membrane and the leakage of the intracellular protein in the photocatalytic antibacterial process. The e-, h+ and O2•- were the active species involved in this process. This study proposed an appropriate photocatalyst for efficient treatment of bacterial contamination.

Evaluation of drug interactions of Saposhnikoviae Radix and its major components with astragaloside IV and paeoniflorin using in vitro and in vivo experiments

Saposhnikoviae Radix (SR) may enhance the pharmacodynamics of Huangqi Chifeng Tang (HQCFT) in the treatment of cerebral infarction according to our previous research, but the underlying mechanism is unknown. Herein, an in vivo pharmacokinetic assay in rats and in vitro MDCK-MDR1 cell assays were used to investigate the possible mechanism of SR, its main components, and its interactions with Astragali Radix (AR) and Paeoniae Radix (PR). An ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC‒MS/MS)-based analytical method for quantifying astragaloside IV (ASIV) and paeoniflorin (PAE) in microdialysis and transport samples was developed. The pharmacokinetic parameters of SR were determined using noncompartmental analyses CCK-8 assays were used to detect the cytotoxicity of ASIV, PAE, cimifugin (CIM), prim-o-glucosylcimifugin (POG) and their combinations. Moreover, drug transport was studied using MDCK-MDR1 cells. Western blotting was performed to measure the protein expression levels of P-GP and MRP1. Claudin-5, ZO-1, and F-actin expression was determined via immunohistochemical staining of MDCK-MDR1 cells. harmacokinetic studies revealed that, compared with those of Huangqi Chifeng Tang-Saposhnikoviae Radix (HQCFT-SR), the Tmax of ASIV increased by 11.11 %, and the MRT0-t and Tmax of PAE increased by 11.19 % and 20 %, respectively, in the HQCFT group. Transport studies revealed that when ASIV was coincubated with 28 μM CIM or POG, the apparent permeability coefficient (Papp) increased by 71.52 % and 50.33 %, respectively. Coincubation of PAE with 120 μM CIM or POG increased the Papp by 87.62 % and 60.95 %, respectively. Moreover, CIM and POG significantly downregulated P-gp and MRP1 (P < 0.05), inhibited the expression of Claudin-5, ZO-1, and F-actin (P < 0.05), and affected intercellular tight junctions (TJs). In conclusion, our study successfully established a selective, sensitive and reproducible UPLC‒MS/MS analytical method to detect drug‒drug interactions between SR, AR and PR in vivo and in vitro, which is beneficial for enhancing the therapeutic efficacies of AR and PR. Moreover, this study provides a theoretical basis for further research on the use of SR as a drug carrier.

Retinal ganglion cell topography and spatial resolving power in the pajama cardinalfish Sphaeramia nematoptera (Bleeker, 1856).

We studied the topography of retinal ganglion cells (GCs) and estimated spatial resolving power (SRP) in the pajama cardinalfish Sphaeramia nematoptera (Bleeker, 1856), a relatively small brightly colored fish inhabiting coral reefs and lagoons in the Western Pacific. S. nematoptera is an active night predator feeding on near-bottom animal plankton and benthos. DAPI staining was used to label nuclei of GCs and non-GCs in the inner plexiform and ganglion cell layers. Non-GCs were distinguished from GCs in Nissl-stained retinal wholemounts based on cell size, shape, and staining intensity. The proportion of displaced amacrine cells (DACs) varied from 15.46 ± 1.12 (visual streak [VS]) to 17.99 ± 1.06% (dorsal periphery) (mean ± S.E.M., N = 5); the respective proportions of glial cells were 6.61 ± 0.84 and 5.89 ± 0.76%. Thus, 76%-78% of cells in the ganglion cell layer and inner plexiform layer were GCs. The minimum spatial coverage of GCs (3600-4600 cells/mm2) was detected in the dorsal and ventral periphery. It gradually increased toward the central retina to form a moderate VS. The maximum GC density (11,400-12,400 cells/mm2) was registered in the central portion of the VS. No pronounced concentric retinal specializations were found. The total number of GCs ranged within 595.2-635.9 × 103. The anatomical spatial resolving power was minimum in the ventral periphery (4.91-5.53 cpd) and maximum in the central portion of the VS (8.47-9.07 cpd). The respective minimum separable angles were 0.18-0.20° and 0.11-0.12°. The relatively high spatial resolving power and presence of the VS in the pajama cardinalfish are in line with its highly visual behavior.

Abstract A006: Spatial localization of Caveolin-1 protein in triple negative breast cancer is related to different molecular features

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive and heterogeneous form of breast cancer with few targeted therapies and clinically used biomarkers. Caveolin-1 (CAV1) is a marker of stromal activation and vascularization. Molecular features of the tumor microenvironment (TME), important for tumor metastasis, may improve precision oncology in TNBC. CAV1 protein levels have previously been reported as a potential biomarker in breast cancer depending on their localization. Therefore, we aimed to investigate CAV1 protein levels in malignant and stromal cells of TNBC and their relation to CAV1 gene expression, clinicopathological factors, and molecular features. Methods: Tumor-specific CAV1 protein levels were evaluated with immunohistochemistry in malignant and stromal cells in tumor tissue microarrays from a cohort of 242 TNBC patients (inclusion 2010-2015, Sweden) from SCAN-B (NCT03758976). The protein levels of CAV1 in malignant and stromal cells were dichotomized as ‘strong’ (1) vs ‘negative to moderate’ (0) The CAV1 categories in malignant and stromal cells, respectively, were combined to create a joint CAV1 status with four categories: malignant/stromal cells, 0/0, 0/1, 1/0, and 1/1. Gene expression profiles from massive parallel paired-end sequencing of mRNA (RNA-seq) were available for all the tumors and from which PAM50 category and TNBC subtype were assigned. CAV1 status in malignant and stromal cells in relation to clinicopathological factors was analyzed using Chi-Square test. CAV1 mRNA expression in relation to CAV1 status in malignant and stromal cells was evaluated using Kruskal-Wallis test. Results: CAV1 status could be evaluated in 231/242 tumors. Strong CAV1 staining in malignant cells was associated with higher histological grade but no axillary lymph node involvement (both P &amp;lt;0.007). In contrast, strong CAV1 staining in stromal cells was associated with lower histological grade but axillary lymph node involvement (both P &amp;lt;0.001). Strong CAV1 staining in stromal cells was also associated with higher age at diagnosis (P =0.025). With regards to PAM50 subtypes, CAV1 in malignant cells was positively associated with the basal subtype while CAV1 in malignant cells was positively associated with the HER2 enriched subtype (P &amp;lt;0.001). Depending on localization, CAV1 expression was associated with different TNBCsubtypes. CAV1 in malignant cells was positively associated with the mesenchymal and negatively associated with the immunomodulatory subtype (P &amp;lt;0.001). CAV1 in stromal cells was positively associated with the luminal androgen (LAR) subtype (P &amp;lt;0.001). Neither CAV1 protein levels in malignant cells nor in stromal cells were correlated with CAV1 gene expression in the tumors, and the combined CAV1 status was not associated with CAV1 gene expression. Conclusion: CAV1 protein levels in malignant cells were related to different molecular features of TNBC compared to CAV1 protein levels in stromal cells. The results suggest that depending on localization, CAV1 protein levels function as two distinct biomarkers. Citation Format: Christopher Godina, Somayeh Khazaei, Mattias Belting, Johan Vallon-Christersson, Björn Nodin, Karin Jirström, Karolin Isaksson, Ana Bosch, Helena Jernström. Spatial localization of Caveolin-1 protein in triple negative breast cancer is related to different molecular features [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A006.

RADA-16-based Self-assembled Peptide Nanofiber Scaffolds Loaded with TGF-β1 Enhance the Chondrogenic Differentiation Potential of BMSCs In vitro.

At present, cartilage repair does not offer ideal efficacy. Fortunately, recent studies have claimed that RADA-16 peptide is an attractive therapeutic strategy for repairing cartilage defects. Therefore, this study tried to explore the effect of RADA-16 loaded with transforming growth factor-beta (TGF-β) 1 on cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs). First, the RADA-16 peptide was synthesized by solid phase peptide, and a well-defined hydrogel was formed by supramolecular peptide self-assembly. Then, TGF-β1 (loading concentration of 10 ng/mL) was loaded into RADA-16, with scanning electron microscopy to observe the morphology of the TGF-β1/RADA-16 hydrogel and detect its related properties. Next, BMSCs were isolated from bone marrow samples and identified. TGF-β1/RADA-16 was co-cultured with L929, BMSCs, and C28/I2 cells, respectively, and the survival and proliferation ability of the cells was determined by live/dead cell staining and MTT assay. Chondrogenic differentiation and sGAG production of BMSCs were determined by Alcian blue staining and Blyscan assay, the expression of cartilage-associated genes by qRT-PCR, and the levels of inflammatory factors by ELISA. As for mechanism investigation, the Smad and ERK/MAPK signaling pathways were detected by western blot. RADA-16 hydrogel exhibited a well-distributed and interconnected porous surface structure, with a loading rate of 91.9% for TGF-β1. The TGF-β1/RADA-16 hydrogel had good release and degradation properties, and had no negative effect on the survival and proliferation ability of BMSCs, L929, and C28/I2 cells. Importantly, TGF-β1/RADA-16 hydrogel significantly accelerated chondrogenic differentiation and sGAG generation in BMSCs, and decreased pro-inflammatory factor production. In addition, the hydrogel also significantly activated the Smad and ERK/MAPK pathways of BMSCs. RADA-16 loaded with TGF-β1 has good biological properties and can enhance the chondrogenic differentiation ability of BMSCs.

High throughput generating stable spheroids with tip-refill wafer.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1%±3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.

Reversible Aptamer Staining, Sorting, and Cleaning of Cells (Clean FACS) with Antidote Oligonucleotide or Nuclease Yields Fully Responsive Cells.

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.

Microglia activity in the human basal ganglia is altered in alcohol use disorder and reversed with remission from alcohol.

Alcohol use disorder (AUD) is characterized by cycles of abuse, withdrawal, and relapse. Neuroadaptations in the basal ganglia are observed in AUD; specifically in the putamen, globus pallidus (GP), and ventral pallidum (VP). These regions are associated with habit formation, drug-seeking behaviors, and reward processing. While previous studies have shown the crucial role of glial cells in drug seeking, it remains unknown whether glial cells in the basal ganglia are altered in AUD. Glial cells in the putamen, GP, and VP were examined in human post-mortem tissue of AUD and alcohol remission cases. Immunohistochemistry was performed to analyze cell count, staining intensity, and morphology of microglia and astrocytes, using markers Iba-1 and GFAP. Morphological analysis revealed a significant decrease in microglia cell size and process retraction, indicating activation or a dystrophic microglia phenotype in individuals with AUD compared to controls. Microglia staining intensity was also higher in the GP and VP in AUD cases, whereas microglia staining intensity and cell size in remission cases were not different to control cases. In contrast, no astrocyte changes were observed in examined brain regions for both AUD and remission cases compared to controls. These results suggest alcohol exposure alters microglia, potentially contributing to dysfunctions in the basal ganglia that maintain addiction, and abstinence from alcohol may reverse microglia changes and associated dysfunctions. Overall, this study further characterizes AUD neuropathology and implicates microglia in the putamen, GP, and VP as a potential target for therapy.

Protective effects of atractylenolide III on oxygen-glucose-deprivation/reperfusion-induced injury in HT22 cells.

Atractylenolide III (ATL III) is a natural bioactive compound, that possesses anti-inflammatory, antioxidant, and neuroprotective properties. However, whether ATL III can protect against neuronal injury induced by cerebral ischemia/reperfusion (I/R) have not yet been studied. This study aimed to investigate the protective effects of ATL III on neuronal injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model in HT22 cells. Establishment of OGD/R model to induce HT22 cell injury in vitro. Cell viability, live-dead cell staining, oxidative stress levels, and pro-inflammatory cytokine levels were detected using kits. Cell apoptosis was observed by flow cytometry, and the expression of Bax, Bcl-2, and Caspase-3 proteins was detected by western blot. ATL III significantly alleviates OGD/R-induced cell injury, as evidenced by the increased cell viability and reduced apoptosis rate. ATL III increased the levels of superoxide dismutase (SOD) and glutathione (GSH), while reducing malondialdehyde (MDA), reactive oxygen species (ROS), and the levels of TNF-α, IL-1β, and IL-6. The protein expression of Bax and Caspase-3 was downregulated, while Bcl-2 expression was upregulated by ATL III. ATL III as a potential therapeutic agent for reducing neuronal injury by mitigating oxidative stress, apoptosis, and inflammation.