Water-soluble Tetrazolium Research Articles

Buprenorphine has a concentration-dependent cytotoxic effect on equine chondrocytes in vitro

To investigate the cytotoxic effects of 2 different concentrations of buprenorphine and compare them with bupivacaine and morphine on healthy equine chondrocytes in vitro. Primary cultured equine articular chondrocytes from 3 healthy adult horses. Chondrocytes were exposed for 0 and 2 hours to the following treatments: media (CON; negative control); bupivacaine at 2.2 mg/mL (BUPI; positive control); morphine at 2.85 mg/mL (MOR); buprenorphine at 0.12 mg/mL (HBUPRE); or buprenorphine at 0.05 mg/mL (LBUPRE). Chondrocyte viability was assessed using live/dead staining, water-soluble tetrazolium salt-8 (WST-8) cytotoxic assay, LDH assay, and flow cytometry. All continuous variables were evaluated with a mixed ANOVA with treatment, time, and their interactions as the fixed effects and each horse as the random effect. Buprenorphine showed a concentration-dependent chondrotoxic effect. The viability of chondrocytes was significantly decreased with exposure to HBUPRE and BUPI compared to CON, MOR, and LBUPRE. Negligible chondrotoxic effects were observed in healthy cultured equine chondrocytes exposed to 0.05 mg/mL of buprenorphine, whereas higher concentrations (0.12 mg/mL) showed a marked cytotoxic effect. Based on these results, low concentrations of buprenorphine appear to be safe for intra-articular administration. Further evaluation of this dose in vivo is needed before recommending its clinical use.

Corydalis decumbens Alleviates the Migration, Phagocytosis, and Inflammatory Response of Macrophages.

The role of Corydalis decumbens (CD) in macrophage activation remains unclear, particularly in the Ras homolog family member A (RhoA) signaling pathway. Therefore, the present study aimed to investigate the effect of CD on the viability, proliferation, morphological changes, migration, phagocytosis, differentiation, and release of inflammatory factors and signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Cell counting kit-8 and water-soluble tetrazolium salt assays were used to evaluate the viability and proliferation of RAW264.7 macrophages. A transwell assay was examined to assess cell migration. The ingestion of lumisphere assay was employed to detect the phagocytic capacity of macrophages. Phalloidin staining was performed to observe morphological changes in the macrophages. An enzyme-linked immunosorbent assay was performed to quantify inflammation-related cytokines in cell culture supernatants. Cellular immunofluorescence and western blotting were adopted to show the expression of inflammation-related factors, biomarkers of M1/M2 subset macrophages, and factors of the RhoA signaling pathway. We found that CD increased the viability and proliferation of RAW264.7 macrophages. CD also impaired the migration and phagocytic capacity of macrophages, induced anti-inflammatory M2 macrophage polarization, such as M2-like morphological changes, and upregulated M2 macrophage biomarkers and anti-inflammatory factors. We also observed that CD inactivated the RhoA signaling pathway. CD mediates the activation of LPS-stimulated macrophages, alleviates the inflammatory responses of macrophages, and activates related signaling pathways induced by LPS.

Early Responses of Neural Cells (U87) to Particulate Matter Exposure: Cell Viability and Cytokine Concentration

Particulate Matter (PM) has a significant impact on human health and increases the risk of respiratory diseases. PM exposure is linked to various illnesses, including cardiovascular diseases, cancer, neurological diseases, and pregnancy complications, and contributes to both mortality and morbidity globally. PM can also influence the autonomic nervous system. The evidence suggests that the likelihood of developing lung cancer continually rises with the rise in the concentration of coarse PM10 (medium aerodynamic diameter less than or equal to 10 μm). However, studies examining the relationship between PM10 exposure and brain cancer or gliomas are scarce. In this study, U87 cells were exposed to PM10 at three different concentrations and the effects were evaluated by analyzing Lactate Dehydrogenase (LDH) activity, Interleukin-6 (IL-6) expression, and cell viability using a water-soluble tetrazolium assay. The IL-6 concentration in U87 cells varied with PM10 concentration and exposure time. PM10 did not influence LDH activity in U87 cells at the tested concentrations. These results provide crucial information for future research aimed at understanding the impact of PM on human disease pathology.

Effects of ‘NEOGEN V.BIOME LIPOSOME™’ on the Increased Expression of COL1A1 and HAS2 mRNA in Human Dermal Fibroblasts

Purpose: This study investigated functional cosmetic agent for anti-wrinkle effects of 'NEOGEN V.BIOME LIPOSOME™', which is a liposome in the form of a proprietary technology including a vitamin C active ingredient, on human dermal fibroblasts (HDFs).Methods: The cytotoxicity of 'NEOGEN V.BIOME LIPOSOME™' was measured by performing a water-soluble tetrazolium salt (WST-1) assay. The relative mRNA expression of collagen (COL1A1 ) and hyaluronan synthase (HAS2) was measured by quantitative real-time PCR (qRT-PCR) assay.Results: HDF cell viability of 'NEOGEN V.BIOME LIPOSOME™' was no cytotoxic effect at concentrations below 0.25%. The expression of COL1A1 and HAS2 mRNA was increased in a concentration-dependent manner compared with the negative control group.Conclusion: “NEOGEN V.BIOME LIPOSOME™“ helps to increase collagen and hyaluronan synthase production by promoting COL1A1 and HAS2 mRNA expression in HDFs; therefore, it is expected to function as an anti-wrinkle cosmetic material.

Cytotoxic effects of Curcuma longa leaves on MCF-7 and HepG2 cells

Curcuma longa (tumeric) is listed in several therapeutic regimens used in the management of cancer. The present study investigated the potential cytotoxic effects of ethanol extract of Curcuma longa (turmeric) leaves on HepG2 (a human hepatocellular carcinoma cell line), and MCF-7 (a human mammary gland adenocarcinoma cell line) cells. The HepG2 and MCF-7 cells were exposed to different concentrations; 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002 μg/ml, of the ethanol extract of C. longa leaves. The Water-Soluble tetrazolium salt (WST-1) assay was used to estimate the cell viability of the cells after which the percentage proliferation was computed. The results of this study indicated that C. longa had cytotoxic effect on the different cell lines, caused a decrease in proliferation and it is more selective to breast cancer cell lines based on the results of WST-1 analysis on the HepG2 and MCF-7 cells. These results suggest that C. longa leaf possesses cytotoxic properties, which can be exploited to develop novel cytotoxic drugs for cancer management. Keywords: Curcuma longa, turmeric, HepG2, MCF-7, WST-1, proliferation, cytotoxic, cancer

Huangqi Decoction, a compound Chinese herbal medicine, inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-β axis

ObjectiveHuangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis. MethodsThe expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting. ResultsLnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment. ConclusionOur study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

Targeting Serine Biosynthesis in Combination with the Modulation of Glutamine Metabolism Enhances Cellular Cytotoxicity in Acute Myeloid Leukemia

Background: Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Gln depletion has emerged as a therapeutic approach for cancers that have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Crisantaspase, an Erwinia asparaginase, has demonstrated promising anti-AML activity in preclinical/clinical models through interference with plasma Gln supply; however, residual leukemia burden was shown in vivo following Gln depletion by a crisantaspase. Following treatment with asparaginases in vitro, we observed a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase AML cell death. To test this, we knocked-out phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, in AML cell lines (MOLM-14 and U937) by CRISPR-cas9 and examined the response of the knockout lines to Gln depletion either through Gln withdrawal from the media or crisantaspase treatment. Methods: Single-cell cloning methods were used on bulk populations of PHGDH CRISPR-cas9 knockout cells to characterize positive knockouts and clones were further confirmed by Sangar sequencing. The impact of Gln depletion and PHGDH knockout on AML cell proliferation/viability was assessed using water soluble tetrazolium (WST-1), trypan blue, and measurement of Lactate Dehydrogenase (LDH) release. Immunoblotting was used to examine the expression of the Ser biosynthesis proteins, PHGDH and phosphoserine aminotransferase (PSAT), and glutathione (GSH) levels were measured via a luminescent-based assay. Results: We determined that short-acting crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values ranging from 0.001 to 0.08 µg/mL. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in cell proliferation/growth, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media (Fig. 1A) as well as following crisantaspase treatment (Fig. 1B). PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from 1.34 to 6.59 picogram/mL, ~250-fold lower compared to the parental lines (Fig. 1C). Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Taken together, these results support dual targeting of Gln and Ser metabolism in AML. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (Ethyl 2-{4-[(1S)-1-[(4,5-dichloro-1,6-dimethyl-1H-indol-2-yl)formamido]-2-hydroxyethyl]benzenesulfonyl}acetate) demonstrated an inhibition of AML cell proliferation in multiple cell lines, with IC50 concentrations ranging between 1.2 to 2.3 µM. Conclusion: Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Polyamidoamine Dendron-Bearing Lipids as Drug-Delivery Excipients.

An amine-terminated polyamidoamine (PAMAM) dendron and two long alkyl groups were designed as a novel drug carrier that possesses an interior for the encapsulation of drugs and a biocompatible surface. We synthesized three dendron-bearing lipids, DL-G1, DL-G2, and DL-G3, which included first, second, and third generation polyamidoamine dendrons, respectively. The synthesized dendrimer encapsulating anticancer drug, 5-fluorouracil (5-FU), was prepared by extraction with chloroform from mixtures of the dendrimers and varying amounts of the drug. In vitro cytotoxicity of PAMAM conjugated di-n-dodecylamine micelles (G1, G2, G3) were analyzed on human gastric adenocarcinoma cells (AGS) by water-soluble tetrazolium-1 (WST-1) cell proliferation assay. Upon exposure to 5-FU loaded micelles, the viability of the cells decreased gradually in all generations. Cytotoxicity increased with increasing generation and reached its highest rate of 69.8 ± 3.2% upon 15 µM 5FU-loaded 25 µM PAMAM DL-3 micelle treatment. These results demonstrate that 5FU-loaded PAMAM conjugated di-n-dodecylamine treatment inhibits the proliferation of AGS cells in a generation-dependent manner.

Chemical Composition of Impatiens textori Miq. Flower Absolute and Its Potential Wound Repair and Anti-Melanogenesis-Promoting Activities in Skin Cells.

Impatiens textori Miq. (ITM; family Balsaminaceae) is a traditional medicinal plant with many biological activities, which include anti-allergic, anti-inflammatory, and anti-pruritic properties. However, it remains to be determined whether ITM affects biological activities in the skin. Thus, we investigated the effects of ITM flower absolute (ITMFAb) extract on the biological activities of skin, especially those related to skin wound repair and whitening. ITMFAb was extracted with hexane, and its composition was determined through GC/MS. The biological activities of ITMFAb on HaCaT keratinocytes and B16BL6 melanoma cells were analyzed using a water-soluble tetrazolium salt, 5-bromo-2'-deoxyuridine incorporation, a Boyden chamber, an ELISA, a sprouting assay, and by immunoblotting. These analyses were performed in a range of ITMFAb concentrations that did not inhibit the viability of the cells (HaCaT, ≤400 µg/mL; B16BL6, ≤200 µg/m). Forty components were identified in ITMFAb. ITMFAb stimulated proliferation, migration, sprout outgrowth, and type I and IV collagen synthesis and upregulated the activations of ERK1/2, JNK, p38 MAPK, and AKT in HaCaT cells. In addition, ITMFAb attenuated the serum-induced proliferation of B16BL6 cells. ITMFAb inhibited melanin synthesis, tyrosinase activity, and expressions of MITF and tyrosinase in α-MSH-exposed B16BL6 cells. These findings indicate that ITMFAb has beneficial effects on wound repairing and whitening-linked responses in the skin and suggest the potential use of ITMFAb as a natural material for the development of skin wound repair and whitening agents.

Feasibility of a Novel 3D Dosimetry System for Commissioning of Single Isocenter Multitarget SRS Treatment

<h3>Purpose/Objective(s)</h3> Verification of Single Isocenter Multitarget SRS treatments (SIM-SRS) is uniquely challenging due to the combination of high dose, steep dose gradients and the requirement for very high spatial accuracy. Here we demonstrate the feasibility of a novel 3D dosimetry system for uniquely comprehensive dosimetry validation and commissioning of this exceptionally challenging technique. <h3>Materials/Methods</h3> The 3D dosimetry system utilizes a new commercially available radiochromic gel dosimeter and a state-of-art in-house telecentric optical-CT readout system DLOS (Duke Large Field of View telecentric Optical CT Scanner). The system was used to quantify the accuracy in 3D and in high resolution (isotropic 1mm) of two SIM-SRS treatments: a simpler two target treatment, and a more complex 5 target treatment. The SIM-SRS plans were created on patient anatomy and then recalculated on a CT of a 15cm diameter dosimeter, with isocenter placed such that each target was located within the dosimeter. Both treatments were delivered to the dosimeters which are gellan gum based radiochromic dosimeters containing a water-soluble tetrazolium salt which reduces into an insoluble formazan dye (with associated color change) under ionizing radiation. Optical-CT readout of the dosimeters pre and post irradiation enabled a high-resolution comprehensive evaluation of the accuracy of delivery. The DLOS is a bi-telecentric system which enables highest accuracy optical-CT readout through strong scatter and stray light rejection. The measured optical density distribution was spatially registered to the dose from the planning system and converted to dose via a calibration curve. Independent verification of treatment accuracy was also performed with a commercial Monte Carlo dose calculation algorithm. <h3>Results</h3> For the 2-target plan, the measured dose agreed with the planning system with a Gamma-Index passing rate of 99.88%, 99.27%, and 92.75%, using a 3%/3mm, 3%/2mm, 5%/1mm criteria, respectively. For the 5-target plan, the Gamma-Index passing rate was 99.59%, 97.35%, and 93.82%, using a 3%/3mm, 3%/2mm, 5%/1mm criteria, respectively. In comparison, the dose calculated using Monte Carlo agreed with the planning system with a Gamma-Index passing rate of 100.0% using 3%/1mm for both the 2 target and 5 target plans. Mean dose for the PTVs of the 2-target plan was within –0.71% and –1.53%. Mean dose for the PTVs of the 5-target plan was within (1.08±0.97)%. <h3>Conclusion</h3> This study demonstrates the feasibility of a new 3D dosimetry tool for verification of advanced radiation treatments. Application to SIM-SRS treatments has demonstrated excellent agreement between measured and predicted dose in the phantom. The combination of DLOS optical-CT with Clearview dosimeters was found to be important in obtaining accurate 3D dosimetry. The method introduced here is anticipated to be applicable to a wide range of clinically challenging treatment techniques.

SIRT1 Protects Against Particulate Matter-Induced Oxidative Stress in Human Corneal and Conjunctival Epithelial Cells.

PurposeSirtuin1 (SIRT1) as a hot therapeutic target for oxidative stress–associated diseases that has been extensively studied. This study aimed to determine the changes in SIRT1 expression in particulate matter (PM)–induced corneal and conjunctival epithelial cell damage and explore potential drugs to reduce PM-associated ocular surface injury.MethodsImmortalized human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs) were exposed to an ambient PM sample. Cytotoxicity was evaluated by water-soluble tetrazolium salt–8 assay. SIRT1 expression was measured by Western blot analysis. Reactive oxygen species (ROS) production, cell apoptosis, mitochondrial function, and cell senescence were assessed by using 2ʹ,7ʹ-dichlorofluorescein diacetate assay, annexin V apoptosis assay, tetramethylrhodamine ethyl ester assay, and senescence β-galactosidase staining, respectively.ResultsPM-induced cytotoxicity of HCECs and HCjECs occurred in a dose-dependent manner. Increased ROS production, as well as decreased SIRT1 expression, were observed in HCECs and HCjECs after 200 µg/mL PM exposure. In addition, PM induced oxidative stress-mediated cellular damage, including cell apoptosis, mitochondrial damage, and cell senescence. Interestingly, SRT1720, a SIRT1 activator, increased SIRT1 expression and decreased ROS production and attenuated PM-induced cell damage in HCECs and HCjECs.ConclusionsThis study determined that SIRT1 was involved in PM-induced oxidative stress in HCECs and HCjECs and found that ROS overproduction may a key factor in PM-induced SIRT1 downregulation. The SIRT1 activator, SRT1720, can effectively upregulate SIRT1 expression and inhibit ROS production, thereby reversing PM-induced cell damage. This study provides a new potential target for clinical treatment of PM-associated ocular surface diseases.

P131 Optimization of MTT assay protocol in Pythium insidiosum zoospores incubated together with neutrophils

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThe tetrazolium-based MTT assay is widely used to assess cell viability. This colorimetric assay measures cell viability through the enzymatic reduction of MTT, a yellow water-soluble tetrazolium dye, to purple-colored formazan crystals. The zoospores of Pythium insidiosum are the infective form causing pythiosis. There remains a lack of standardized protocol for P. insidiosum zoospore viability assay. Here, we optimized the MTT assay to assess the activity of neutrophils on zoospores.MethodsNeutrophils and zoospores were incubated for 2 h, 4 h, and overnight at 37°C. Neutrophils were lysed in water (pH = 11), MTT was added to each well and incubated at 37°C for 2 h. Then, SDS-HCl solution was added to each well, and absorbance at 570 nm was measured using a microplate reader. The percentage of viable zoospores was then determined.ResultsThe viable zoospores metabolized MTT to insoluble purple formazan product. The zoospores germinated into hyphae when incubated for ˃2 h. Because of the rapid germination of zoospores into hyphae, reliable results would be obtained with an optimum incubation time of 2 h for the zoospores.ConclusionThe simplicity and low cost of the assay make it a valuable tool for zoospore viability studies, with a focus on immunology and immunotherapy.

Comparative chemical properties, bioactivity, and cytotoxicity of resin-modified calcium silicate-based pulp capping materials on human dental pulp stem cells.

This study investigated the cytotoxicity, the residual monomer release, degree of conversion (DC), calcium ion (Ca2+) release, and crystal structure of TheraCal PT (ThPT) by comparison with TheraCal LC (ThLC) and mineral trioxide aggregate (MTA). The cytotoxicity of the cured materials was evaluated on human dental pulp stem cells (hDPSCs) isolated from third molars by the water-soluble tetrazolium salt (WST-1) method. The monomer release and DC of the resin-containing materials were analyzed by high-performance liquid chromatography (HPLC) and Fourier transform infrared (FTIR), respectively. The chemical composition and Ca2+ release of the materials were determined by scanning electronic microscopy-energy-dispersive spectroscopy (SEM-EDS), X-ray diffractometry (XRD), and inductively coupled plasma-optical emission spectroscopy (ICP-OES), respectively. Statistical differences were evaluated with one-way ANOVA, repeated measure ANOVA, and the Tukey test (p < 0.05). MTA showed significantly lower cytotoxicity than either ThLC or ThPT after 1, 3, and 7days (p < 0.05). TEGDMA release of ThPT is significantly higher than ThLC (p < 0.05). All materials showed calcium Ca2+ release, with MTA significantly higher than the others (p < 0.05). MTA showed low cytotoxicity and high Ca2+ release compared to ThLC and ThPT. The cytotoxicity and residual monomer release of ThLC and ThPT may raise concerns about the viability of hDPSCs. Further investigations with the use of in vivo research models are required to validate in vitro bioactivity properties and the potential adverse biological effects of ThLC and ThPT on hDPSCs.

Isolation of Aloe saponaria-Derived Extracellular Vesicles and Investigation of Their Potential for Chronic Wound Healing.

A chronic wound is caused by a failure to progress through the normal phases of wound repair in an orderly and timely manner. To induce skin regeneration while inhibiting chronic inflammation, numerous natural products, and in particular, plant-derived biomaterials, have been developed. Aloe saponaria, is known to contain flavonoid and phenolic acid compounds with anti-oxidative and anti-inflammatory properties. Here, we isolated extracellular vesicles (EVs) from Aloe saponaria by polyethylene glycol (PEG)-based precipitation and investigated their potential as a therapeutic for chronic wound healing. The Aloe saponaria-derived EVs (AS-EVs) showed no significant cytotoxicity on several cell types, despite a high level of intracellular uptake. When lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were treated with AS-EVs, significant reductions in the expression of pro-inflammatory genes, such as interleukin-6 and interleukin-1β, were observed. Proliferation and migration of human dermal fibroblasts, as determined by the water-soluble tetrazolium salt-8 and transwell migration assay, respectively, were shown to be promoted by treatment with AS-EVs. It was also demonstrated that AS-EVs enhanced tube formation in human umbilical vein endothelial cells, indicating a stimulatory activity on angiogenesis; one of the crucial steps for effective wound healing. Collectively, our results suggest the potential of AS-EVs as a natural therapeutic for chronic wound healing.

In vitro cytocompatibility and antibacterial studies on biodegradable Zn alloys supplemented by a critical assessment of direct contact cytotoxicity assay.

In vitro cytotoxicity assessment is indispensable in developing new biodegradable implant materials. Zn, which demonstrates an ideal corrosion rate between Mg- and Fe-based alloys, has been reported to have excellent in vivo biocompatibility. Therefore, modifications aimed at improving Zn's mechanical properties should not degrade its biological response. As sufficient strength, ductility and corrosion behavior required of load-bearing implants has been obtained in plastically deformed Zn-3Ag-0.5Mg, the effect of simultaneous Ag and Mg additions on in vitro cytocompatibility and antibacterial properties was studied, in relation to Zn and Zn-3Ag. Direct cell culture on samples and indirect extract-based tests showed almost no significant differences between the tested Zn-based materials. The diluted extracts of Zn, Zn-3Ag, and Zn-3Ag-0.5Mg showed no cytotoxicity toward MG-63 cells at a concentration of ≤12.5%. The cytotoxic effect was observed only at high Zn2+ ion concentrations and when in direct contact with metallic samples. The highest LD50 (lethal dose killing 50% of cells) of 13.4mg/L of Zn2+ ions were determined for the Zn-3Ag-0.5Mg. Similar antibacterial activity against Escherichia coli and Staphylococcus aureus was observed for Zn and Zn alloys, so the effect is attributed mainly to the released Zn2+ ions exhibiting bactericidal properties. Most importantly, our experiments indicated the limitations of water-soluble tetrazolium salt-based cytotoxicity assays for direct tests on Zn-based materials. The discrepancies between the WST-8 assay and SEM observations are attributed to the interference of Zn2+ ions with tetrazolium salt, therefore favoring its transformation into formazan, giving false cell viability quantitative results.

Green Synthesis of Magnetic Ferrites (Fe3O4, CoFe2O4, and NiFe2O4) Stabilized by Aloe Vera Extract for Cancer Hyperthermia Activities

The magnetic nanoferrites (Fe <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> , CoFe <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> , and NiFe <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sub> ) were synthesized via hydrothermal method using an aloe vera plant extract solution. Magnetic nanoferrites were characterized using X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM), field emission electron microscope (FESEM), transmission electron microscopy (TEM), and X-ray photon spectroscopy (XPS). Cytotoxicity was obtained using the water-soluble tetrazolium salt (WST) assay. X-ray diffraction analysis also demonstrated that the size range of the nanoferrites exhibited average diameters of 6–28 nm and also exhibited high crystallinity. Results showed that all the metallic nanoparticles (NPs) are magnetic with saturation values of Fe = 52 emu/g, Co = 33 emu/g, and Ni = 20.43 emu/g. The magnetic nanoferrites exhibited a high cell viability rate at high concentrations and as shown from the optical images of the cells signifies their nontoxic and biocompatibility property. From SEM and TEM, the spherical structure was noticeable and the particle size is also in accordance with XRD. The chemical states for each NP also confirmed the successful synthesis of each nanoferrite. Not to mention, the heating capabilities of the nanoferrites were investigated and showed significant temperature increase of 6 °C–8 °C and acceptable specific absorption rate (SAR) values. Interestingly, the results assure that the materials would be suitable as magnetic mediators for magnetic hyperthermia application for cancer treatment.

Soluble guanylate cyclase stimulation fosters angiogenesis and blunts myofibroblast-like features of systemic sclerosis endothelial cells.

In SSc, angiogenesis impairment advances in parallel with the development of fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-mesenchymal transition (EndoMT). Soluble guanylate cyclase (sGC) stimulation has shown antifibrotic effects in SSc skin fibroblasts and mouse models. Here, we investigated the effects of pharmacological sGC stimulation on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-MVECs). To determine whether sGC stimulation affected cell viability/proliferation, SSc-MVECs and healthy dermal MVECs (H-MVECs) were challenged with the sGC stimulator (sGCS) MK-2947 and assayed by annexin V/propidium iodide flow cytometry and the water-soluble tetrazolium salt (WST-1) assay. To study angiogenesis and EndoMT, MK-2947-treated SSc-MVECs were subjected to wound healing and capillary morphogenesis assays and analysed for the expression of endothelial/myofibroblast markers and contractile ability. MK-2947 treatment did not affect H-MVEC viability/proliferation, while it significantly increased SSc-MVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-MVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in the expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 mesenchymal markers was also found in MK-2947-treated SSc-MVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-MVECs to contract collagen gels and reduced phosphorylated-extracellular signal-regulated kinases 1 and 2 protein levels. These findings demonstrate for the first time that pharmacological sGC stimulation effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-MVECs, thus providing new evidence for repurposing sGCSs for SSc.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

BackgroundTyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.MethodsKINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic–pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.ResultsIn mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.ConclusionsTaken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial registrationClinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Genotoxicity of Particles From Grinded Plastic Items in Caco-2 and HepG2 Cells.

Large plastic litters degrade in the environment to micro- and nanoplastics, which may then enter the food chain and lead to human exposure by ingestion. The present study explored ways to obtain nanoplastic particles from real-life food containers. The first set of experiments gave rise to polypropylene nanoplastic suspensions with a hydrodynamic particle size range between 100 and 600 nm, whereas the same grinding process of polyethylene terephthalate (PET) produced suspensions of particles with a primary size between 100 and 300 nm. The exposure did not cause cytotoxicity measured by the lactate dehydrogenase (LDH) and water soluble tetrazolium 1 (WST-1) assays in Caco-2 and HepG2 cells. Nanoplastics of transparent PET food containers produced a modest concentration-dependent increase in DNA strand breaks, measured by the alkaline comet assay [net induction of 0.28 lesions/106 bp at the highest concentration (95% CI: 0.04; 0.51 lesions/106 base pair)]. The exposure to nanoplastics from transparent polypropylene food containers was also positively associated with DNA strand breaks [i.e., net induction of 0.10 lesions/106 base pair (95% CI: −0.04; 0.23 lesions/106 base pair)] at the highest concentration. Nanoplastics from grinding of black colored PET food containers demonstrated no effect on HepG2 and Caco-2 cells in terms of cytotoxicity, reactive oxygen species production or changes in cell cycle distribution. The net induction of DNA strand breaks was 0.43 lesions/106 bp (95% CI: 0.09; 0.78 lesions/106 bp) at the highest concentration of nanoplastics from black PET food containers. Collectively, the results indicate that exposure to nanoplastics from real-life consumer products can cause genotoxicity in cell cultures.

Vancomycin Containing PDLLA and PLGA/β-TCP Inhibit Biofilm Formation but Do Not Stimulate Osteogenic Transformation of Human Mesenchymal Stem Cells.

AimsChronic osteomyelitis, including implant-related prosthetic joint infection, is extremely difficult to cure. We develop vancomycin containing release systems from poly(d,l-lactide) (PDLLA) and poly(d,l-lactide-co-glycolide) (PLGA) composites with beta-tricalcium phosphate (β-TCP) to treat methicillin-resistant Staphylococcus aureus osteomyelitis. We ask whether vancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites will prevent early biofilm formation, allow cell proliferation and osteogenic differentiation, and stimulate osteogenic signaling molecules in the absence of an osteogenic medium.MethodsComposites were produced and characterized with scanning electron microscopy. In vitro vancomycin release was assessed for 6 weeks. Biofilm prevention was calculated by crystal violet staining. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and osteosarcoma cell (SaOS-2) proliferation and differentiation were assessed with water soluble tetrazolium salt and alkaline phosphatase (ALP) staining. Real-time quantitative polymerase chain reaction defined osteogenic signaling molecules for hBM-MSCs.ResultsTotally, 3.1 ± 0.2 mg and 3.4 ± 0.4 mg vancomycin released from PDLLA/β-TCP and the PLGA/β-TCP composites, respectively, and inhibited early biofilm formation. hBM-MSCs and SaOS-2 cells proliferated on the composites and stimulated ALP activity of cells. Runt-related transcription factor 2 (RUNX2) and SRY-Box transcription Factor 9 (SOX9) expressions were, however, lower with composites when compared with control.ConclusionVancomycin containing PDLLA/β-TCP and PLGA/β-TCP composites inhibited early biofilm formation and proliferated and differentiated hBM-MSCs and SaOS-2 cells, but osteogenesis-related RUNX2 and SOX9 transcription factors were not strongly expressed in the absence of an osteogenic medium for 14 days.