Trypan Blue Dye Exclusion Test Research Articles

Cancer Stem Cell and Epithelial Mesenchymal Transition (EMT) in Canine Transmissible Venereal Tumour- A Preliminary Study

Cancer Stem Cell and Epithelial Mesenchymal Transition (EMT) in Canine Transmissible Venereal Tumour- A Preliminary Study

Anti-blastocystis activity of hop extracts in vitro

Blastocystis sp. are the most common, unicellular, anaerobic parasites of the intestinal tract of many animal and human species that can cause various digestive diseases. Metronidazole has long been used as a first-line treatment for blastocystosis, but recent clinical and in vitro studies have demonstrated its low efficacy against Blastocystis sp. The aim of this study was to determine the in vitro sensitivity of Blastocystis sp. clinical isolates to carbonic acid hop extracts and alcohol hop extracts in comparison with metronidazole. Five cultures of Blastocystis sp. were isolated from faecal samples from patients with irritable bowel syndrome with predominant diarrhoea (IBS-D, Rome IV). The parasites were identified by microscopy of faecal smears permanently stained with trichrome, Wheatley’s modification and Heidenhain’s iron-haematoxylin. Blastocystis sp. was cultivated at 37°C under anaerobic conditions on RPMI-1640 with antibiotics and horse serum, taking into account their growth characteristics. To detect the anti­blastocystic activity hop extracts and metronidazole were tested in the range from 1000 μg/ml to 1 μg/ml.The presence and number of viable Blastocystis sp. cells were determined after 24, 48, 72 and 96 hours. Blastocystis sp. cells were counted in a hemocytometer using the trypan blue dye exclusion test. All experiments were performed in triplicate. According to the results of in vitro sensitivity of 5 clinical isolates of Blastocystis sp. to the action of carbonic acid hop extract, alcohoicl hop extract and metronidazole, a direct positive pattern in the dose-response and contact time-response effects was established. Alcoholic hop extract showed the highest level of antiblastocystic activity with indicators (for 72-hour parasite cultures) of the minimum inhibitory concentration (which inhibits the parasite cultures growth by 50%) (2.8±0,8) μg/ml and the minimum lethal concentration (which destroys parasite cells by 100%) – 8 μg/ml, being 2.4 and 4.5 times lower than the minimum inhibitory concentration and 8 and 62.5 times lower, than the minimum lethal concentration for hop extract and metronidazole, respectively (p<0.05). It has been shown that alcoholic hop extract (≥16 μg/ml) causes gradual morphological changes in Blastocystis sp. cells, leading to their complete destruction.In contrast to metronidazole, subinhibitory concentrations of hop extract (<2 μg/ml) do not stimulate the proliferation of Blastocystis sp. cells in vitro.

Titanium Surfaces with a Laser-Produced Microchannel Structure Enhance Pre-Osteoblast Proliferation, Maturation, and Extracellular Mineralization In Vitro.

The clinical success of dental titanium implants is profoundly linked to implant stability and osseointegration, which comprises pre-osteoblast proliferation, osteogenic differentiation, and extracellular mineralization. Because of the bio-inert nature of titanium, surface processing using subtractive or additive methods enhances osseointegration ability but limits the benefit due to accompanying surface contamination. By contrast, laser processing methods increase the roughness of the implant surface without contamination. However, the effects of laser-mediated distinct surface structures on the osteointegration level of osteoblasts are controversial. The role of a titanium surface with a laser-mediated microchannel structure in pre-osteoblast maturation remains unclear. This study aimed to elucidate the effect of laser-produced microchannels on pre-osteoblast maturation. Pre-osteoblast human embryonic palatal mesenchymal cells were seeded on a titanium plate treated with grinding (G), sandblasting with large grit and acid etching (SLA), or laser irradiation (L) for 3-18 days. The proliferation and morphology of pre-osteoblasts were evaluated using a Trypan Blue dye exclusion test and fluorescence microscopy. The mRNA expression, protein expression, and protein secretion of osteogenic differentiation markers in pre-osteoblasts were evaluated using reverse transcriptase quantitative polymerase chain reaction, a Western blot assay, and a multiplex assay, respectively. The extracellular calcium precipitation of pre-osteoblast was measured using Alizarin red S staining. Compared to G- and SLA-treated titanium surfaces, the laser-produced microchannel surfaces enhanced pre-osteoblast proliferation, the expression/secretion of osteogenic differentiation markers, and extracellular calcium precipitation. Laser-treated titanium implants may enhance the pre-osteoblast maturation process and provide extra benefits in clinical application.

78 Improvement of Cell Viability in Temporary Storage of Split Thickness Skin Grafts

Abstract Introduction Autologous split thickness skin grafts (STSGs) are the preferred donor tissue for restoration of large tissue deficits. Although grafting should be done at the time of donor harvest, various factors necessitate tissue storage, traditionally in saline solution (SS) at 4oC. However, STSGs often expire before grafting in these conditions. Patients then require further excision procedures or die due to inadequate skin coverage. For patients’ wellbeing, establishing a new storage protocol is imperative. This study sought to determine an effective storage media for prolongation of graft viability for subsequent incorporation into excision and grafting practice. Methods Following patient consent, excess tissue >1cm2 was collected after grafting procedures. Tissue was stored at 4oC in RPMI, DMEM, and SS with penicillin/streptomycin. Samples were evaluated at 2, 5, 7, and 14 days. Cell viability was measured by Trypan blue dye exclusion test in an automated cell counter yielding percentage of viable keratinocytes (PVK). An unbiased pathologist assessed tissue integrity by epidermal-dermal junction, perinuclear keratinocyte halos, and collagen organization with higher scores denoting deterioration. Data were evaluated by ANOVA and Effect size. Results A total of 155 sets of two specimens each were collected from 13 patients. Mean PVK values at day 2, 5, 7, and 14: SS (57.38, 36.96, 38.38, 27.50), RPMI (69.46, 69.92, 70.33, 62.67), DMEM (63.17, 53.50, 54.83, 37.92). By pairwise comparison, RPMI preserved PVK better than SS at days 5, 7, and 14 (p = 0.001 for each day). DMEM preserved PVK better than SS when comparing all days (p = 0.011) but failed to produce a statistically significant difference at any specific day. RPMI preserved PVK greater than DMEM each day. However, statistically significant differences were only revealed at day 14 (p = 0.017) and when comparing all days (p < 0.001). Effect size was large for overall group differences of PVK at day 5, 7, and 14 (Cohen’s f = 0.72, 0.73, and 0.75). Tissue integrity scores at day 2, 5, 7, and 14: SS (0.69, 1.0, 1.38, 2.0), RPMI (0.54, 0.92, 0.85, 1.54), DMEM (0.77, 0.92, 0.77, 1.15). Although culture media preserved tissue integrity greater than SS, no statistically significant differences were found. Conclusions RPMI preserved cell viability better than DMEM and SS. This suggests that STSGs stored in RPMI may prolong the time of which tissue could be stored and successfully amalgamated with skin. Applicability of Research to Practice Storing tissue in RPMI could benefit burn patients by decreasing tissue disposal therefore reducing future donor site area or eliminating repetitious excision procedures entirely. These effects decrease inpatient days and healthcare costs. Most importantly, successful tissue storage will save patients’ lives, especially those of large surface area burns who lack sufficient donor sites.

Myb Repression Mediates Stat5b-knockdown-induced Apoptosis and Inhibits Proliferation of Glioblastoma Stem Cells.

Glioblastoma is the most common and aggressive malignant brain tumor in adults, and glioblastoma stem cells (GSCs) contribute to treatment resistance and recurrence. Inhibition of Stat5b in GSCs suppresses cell proliferation and induces apoptosis. Herein, we investigated the mechanisms of growth inhibition by Stat5b knockdown (KD) in GSCs. GSCs were established from a murine glioblastoma model in which shRNA-p53 and EGFR/Ras mutants were induced in vivo using a Sleeping Beauty transposon system. Microarray analyses were performed on Stat5b-KD GSCs to identify genes that are differentially expressed downstream of Stat5b. RT-qPCR and western blot analyses were used to determine Myb levels in GSCs. Myb-overexpressing GSCs were induced by electroporation. Proliferation and apoptosis were evaluated by a trypan blue dye exclusion test and annexin-V staining, respectively. MYB, which is involved in the Wnt pathway, was identified as a novel gene whose expression was down-regulated by Stat5b-KD in GSCs. Both MYB mRNA and protein levels were down-regulated by Stat5b-KD. Overexpression of Myb rescued cell proliferation that was suppressed by Stat5b-KD. Furthermore, Stat5b-KD-induced apoptosis in GSCs was significantly inhibited by Myb overexpression. Down-regulation of Myb mediates Stat5b-KD-induced inhibition of proliferation and induction of apoptosis in GSCs. This may represent a promising novel therapeutic strategy against glioblastoma.

ONCOLYTIC ACTIVITY OF HUMAN ORTHOPNEUMOVIRUS IN CANCER CELL LINES.

This study was designed to elucidate the possible role of hOPV in the modulation of cell growth and apoptosis in cancer cell lines including human epidermoid carcinoma (HEp-2), lung epithelial cell line (A549), and breast cancer cell line (MCF-7). The oncolytic activity of hOPV on cancer cells was studiedin vitro. The virus titersweredeterminedbytissue culture infectious dose (TCID50/mL) in A549cell. The cytotoxic effect of the virus onHEp-2, A549, and MCF-7was determined using MTT and trypan blue dyeexclusion test assays. hOPV in the infected cells was detected using real-time reverse transcription polymerase chain reaction(rRT-PCR) and indirect immunofluorescence (IIF) assays. Therelative expression of apoptosis-related genes (CASP-3, -8, -9, Bax, Bcl-2, Bcl-XL, TP53, P21) during virus infection was estimated using rRT-PCR assay in comparison with the house-keeping gene (GAPDH). hOPV infection inhibited the growth of HEp-2, A549, and MCF-7cells in a dose-and time-dependent manner. At a multiplicity of infection (MOI) of 5, hOPV reduced the viability of A549cells to about 16%, HEp-2to 22%, and MCF-7to 28% (p = 0.001), while no significant inhibitory effect was observed when cells were infected at MOI of 1and 2. hOPV mRNA and antigens were detected in infected HEp-2, A549, and MCF-7cells by RT-PCR and IIF. Upon hOPV infection, expression of CASP-3, -8, -9, as well as Bax, TP53, and p21mRNA increased while expression of Bcl-2, Bcl-xL anti-apoptotic genes decreased. In hOPV-infected A549cells, the fold increase of CASP-8and CASP-9, Bax, TP53, and P21expression exceeded significantly compared to that in HEp-2or MCF-7cells. Ourresults provide evidence that hOPV could be a potential candidate for oncolytic virotherapy.

Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-β1-induced fibrotic response in normal human dermal fibroblasts

BackgroundAbnormal and deregulated skin wound healing associated with prolonged inflammation may result in dermal fibrosis. Since the current therapeutic strategies revealed unsatisfactory, the investigation of alternative approaches such as those based on the use of specific probiotic strains could provide promising therapeutic options. In this study, we aimed to evaluate whether the lysate from S. thermophilus could antagonize the fibrogenic effects of TGF-β1 in normal human dermal fibroblasts (NHDF).MethodsNHDF were exposed to TGF-β1 to establish a fibrotic phenotype. Proliferation rate and cell number were measured using the IncuCyte® Live Cell Imager system and the trypan blue dye exclusion test. Phenoconversion markers (α-SMA and fibronectin) and collagen I levels were assessed by western blot and immunofluorescence. The mRNA levels of TGF-β1 were evaluated by RT-PCR. The Smad2/3 phosphorylation level as well as β-catenin and PPARγ expression, were assessed by western blot. The cell contractility function and migration of NHDF were studied using collagen gel retraction assay, and scratch wound healing assay, respectively. The effects of S. thermophilus lysate, alone or combined with TGF-β1, were evaluated on all of the above-listed parameters and markers associated with TGF-β1-induced fibrotic phenotype.ResultsExposure to the S. thermophilus lysate significantly reduced the key mediators and events involved in the abnormal activation of myofibroblasts by TGF-β1 within the fibrotic profile. The S. thermophilus treatment significantly reduced cell proliferation, migration, and myo-differentiation. In addition, the treatment with probiotic lysate reduced the α-SMA, fibronectin, collagen-I expression levels, and affected the collagen contraction ability of activated dermal fibroblasts. Moreover, the probiotic targeted the TGF-β1 signaling, reducing Smad2/3 activation, TGF-β1 mRNA level, and β-catenin expression through the upregulation of PPARγ.ConclusionThis is the first report showing that S. thermophilus lysate had a remarkable anti-fibrotic effect in TGF-β1-activated NHDF by inhibiting Smad signaling. Notably, the probiotic was able to reduce β-catenin and increase PPARγ levels. The findings support our point that S. thermophilus may help prevent or treat hypertrophic scarring and keloids.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Synthesis of Sugar and Nucleoside Analogs and Evaluation of Their Anticancer and Analgesic Potentials.

Chemical modification of sugars and nucleosides has a long history of producing compounds with improved selectivity and efficacy. In this study, several modified sugars (2–3) and ribonucleoside analogs (4–8) have been synthesized from α-d-glucose in a total of 21 steps. The compounds were tested for peripheral anti-nociceptive characteristics in the acetic acid-induced writhing assay in mice, where compounds 2, 7, and 8 showed a significant reduction in the number of writhes by 56%, 62%, and 63%, respectively. The compounds were also tested for their cytotoxic potential against human HeLa cell line via trypan blue dye exclusion test followed by cell counting kit-8 (CCK-8) assay. Compound 6 demonstrated significant cytotoxic activity with an IC50 value of 54 µg/mL. Molecular docking simulations revealed that compounds 2, 7, and 8 had a comparable binding affinity to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. Additionally, the bridged nucleoside analogs 7 and 8 potently inhibited adenosine kinase enzyme as well, which indicates an alternate mechanistic pathway behind their anti-nociceptive action. Cytotoxic compound 6 demonstrated strong docking with cancer drug targets human cytidine deaminase, proto-oncogene tyrosine-protein kinase Src, human thymidine kinase 1, human thymidylate synthase, and human adenosine deaminase 2. This is the first ever reporting of the synthesis and analgesic property of compound 8 and the cytotoxic potential of compound 6.

JCI‑20679 suppresses the proliferation of glioblastoma stem cells by activating AMPK and decreasing NFATc2 expression levels.

The prognosis of glioblastoma, which is the most frequent type of adult‑onset malignant brain tumor, is extremely poor. Therefore, novel therapeutic strategies are needed. Previous studies report that JCI‑20679, which is synthesized based on the structure of naturally occurring acetogenin, inhibits mitochondrial complex I and suppresses the growth of various types of cancer cells. However, the efficacy of JCI‑20679 on glioblastoma stem cells (GSCs) is unknown. The present study demonstrated that JCI‑20679 inhibited the growth of GSCs derived from a transposon system‑mediated murine glioblastoma model more efficiently compared with the growth of differentiation‑induced adherent cells, as determined by a trypan blue staining dye exclusion test. The inhibition of proliferation was accompanied by the blockade of cell‑cycle entry into the S‑phase, as assessed by a BrdU incorporation assay. JCI‑20679 decreased the mitochondrial membrane potential, suppressed the oxygen consumption rate and increased mitochondrial reactive oxygen species generation, indicating that JCI‑20679 inhibited mitochondrial activity. The mitochondrial inhibition was revealed to increase phosphorylated (phospho)‑AMPKα levels and decrease nuclear factor of activated T‑cells 2 (NFATc2) expression, and was accompanied by a decrease in calcineurin phosphatase activity. Depletion of phospho‑AMPKα by knockdown of AMPKβ recovered the JCI‑20679‑mediated decrease in NFATc2 expression levels, as determined by western blotting and reverse transcription‑quantitative PCR analysis. Overexpression of NFATc2 recovered the JCI‑20679‑mediated suppression of proliferation, as determined by a trypan blue staining dye exclusion test. These results suggest that JCI‑20679 inhibited mitochondrial oxidative phosphorylation, which activated AMPK and reduced NFATc2 expression levels. Moreover, systemic administration of JCI‑20679 extended the event‑free survival rate in a mouse model transplanted with GSCs. Overall, these results suggested that JCI‑20679 is a potential novel therapeutic agent against glioblastoma.

Abstract 1427: Sensitizing HL60 acute myeloid leukemia cells to decitabine with pterostilbene

Abstract Introduction. Decitabine (DAC), is a DNA hypomethylating agent that has been shown to be potent towards leukemia cells at low doses. However, many patients develop resistance to the drug due to dysregulation of genes associated with responsiveness to DAC-induced DNA demethylation. To this regard, it has been reported that downregulation of TET2, one of the demethylating enzymes, increases sensitivity to DAC in acute myeloid leukemia (AML) cells. Remarkably, dietary bioactive compounds such as pterostilbene (PTS), with a stilbenoid ring structure, have previously been shown to mediate changes in gene expression through regulation of the epigenetic machinery in cancer models. Thus, we sought to test the impact of treatment of leukemia cells with PTS to augment expression of genes involved in the responsiveness of low-dose DAC in AML cells. Methods. HL60, an AML cell line, was either treated in combination with PTS (1.0 µM) and DAC (200 nM) or pre-treated with different concentrations of PTS (0 µM, 0.5 µM, 1.0 µM, 2 µM) for four days followed by DAC treatment (100 nM) for three days. Cell growth was assessed via trypan blue dye exclusion test and comparisons were made between combination treatment and pre-treatment with PTS. qPCR was performed in triplicates for each assay to assess expression of selected genes. Results. Treatment of HL60 cells with 200 nM DAC alone led to a 60% decrease in cell growth compared to control (treated with ethanol as a vehicle). After the combined treatment with PTS, cell growth decreased by further 27% compared to DAC alone. In order to decipher if this decrease in cell growth was due to the effect of PTS sensitizing cells to DAC, the cells were first pre-treated with PTS for four days and subsequently treated with DAC for three days. Compared to control (ethanol pre-treated cells), growth of cells treated with 100 nM DAC alone was decreased by about 37%, while growth of cells pre-treated with 0.5 µM, 1.0 µM, and 2.0 µM PTS followed by 100 nM DAC exposure was further reduced by 13%, 14.5%, and 30%, respectively. This suggests that PTS-pre-treated cells are more sensitive to DAC. Interestingly, DAC alone at 200 nM led to an increase in expression of TET2 (p<0.01) while PTS at 1.0 µM alone led a decrease (p<0.01) compared to control. However, when PTS and DAC were combined, TET2 levels increased significantly (p<0.01) compared to both control and DAC alone, despite its better ability to inhibit growth. In order to explore this further, we will proceed with investigating the effects of pre-treatment with PTS on TET2 expression in HL60 cells. Conclusion. Treatment of HL60 cells with PTS results in improved sensitivity of cells to low doses of DAC, possibly due to modulating TET2 expression. Citation Format: Cayla Boycott, Barbara Stefanska. Sensitizing HL60 acute myeloid leukemia cells to decitabine with pterostilbene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1427.

The anti-tumor efficacy of 20(S)-protopanaxadiol, an active metabolite of ginseng, according to fasting on hepatocellular carcinoma

Background20(S)-protopanaxadiol (20(S)-PPD), one of the main active metabolites of ginseng, performs a broad spectrum of anti-tumor effects. Our aims are to search out new strategies to enhance anti-tumor effects of natural products, including 20(S)-PPD. In recent years, fasting has been shown to be multi-functional on tumor progression. Here, the effects of fasting combined with 20(S)-PPD on hepatocellular carcinoma growth, apoptosis, migration, invasion and cell cycle were explored. MethodsCCK-8 assay, trypan blue dye exclusion test, imagings photographed by HoloMonitorTM M4, transwell assay and flow cytometry assay were performed for functional analyses on cell proliferation, morphology, migration, invasion, apoptosis, necrosis and cell cycle. The expressions of genes on protein levels were tested by western blot. Tumor-bearing mice were used to evaluate the effects of intermittent fasting combined with 20(S)-PPD. ResultsWe firstly confirmed that fasting-mimicking increased the anti-proliferation effect of 20(S)-PPD in human HepG2 cells in vitro. In fasting-mimicking culturing medium, the apoptosis and necrosis induced by 20(S)-PPD increased and more cells were arrested at G0-G1 phase. Meanwhile, invasion and migration of cells were decreased by down-regulating the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in fasting-mimicking medium. Furthermore, the in vivo study confirmed that intermittent fasting enhanced the tumor growth inhibition of 20(S)-PPD in H22 tumor-bearing mice without obvious side effects. ConclusionFasting significantly sensitized HCC cells to 20(S)-PPD in vivo and in vitro. These data indicated that dietary restriction can be one of the potential strategies of chinese medicine or its active metabolites against hepatocellular carcinoma.

Soluble Fraction from Lysate of a High Concentration Multi-Strain Probiotic Formulation Inhibits TGF-β1-Induced Intestinal Fibrosis on CCD-18Co Cells.

Fibrosis is a severe complication of chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Current strategies are not fully effective in treating fibrosis; therefore, innovative anti-fibrotic approaches are urgently needed. TGF-β1 plays a central role in the fibrotic process by inducing myofibroblast differentiation and excessive extracellular matrix (ECM) protein deposition. Here, we explored the potential anti-fibrotic impact of two high concentration multi-strain probiotic formulations on TGF-β1-activated human intestinal colonic myofibroblast CCD-18Co. Human colonic fibroblast CCD-18Co cells were cultured in the presence of TGF-β1 to develop a fibrotic phenotype. Cell viability and growth were measured using the Trypan Blue dye exclusion test. The collagen-I, α-SMA, and pSmad2/3 expression levels were evaluated by Western blot analysis. Fibrosis markers were also analyzed by immunofluorescence and microscopy. The levels of TGF-β1 in the culture medium were assessed by ELISA. The effects of commercially available probiotic products VSL#3® and Vivomixx® were evaluated as the soluble fraction of bacterial lysates. The results suggested that the soluble fraction of Vivomixx® formulation, but not VSL#3®, was able to antagonize the pro-fibrotic effects of TGF-β1 on CCD-18Co cells, being able to prevent all of the cellular and molecular parameters that are related to the fibrotic phenotype. The mechanism underlying the observed effect appeared to be associated with inhibition of the TGF-β1/Smad signaling pathway. To our knowledge, this study provides the first experimental evidence that Vivomixx® could be considered to be a promising candidate against intestinal fibrosis, being able to antagonize TGF-β1 pro-fibrotic effects. The differences that were observed in our fibrosis model between the two probiotics used could be attributable to the different number of strains in different proportions.

Assessment of Therapeutic Ultrasound Effect on Cell Viability, Osmotic Integrity, and Dielectric Behavior

Ultrasound (US) is a sound wave with frequency beyond the human hearing limit (20 kHz). US is relatively available, simple and energy saving. As a result it became hence an emerging technology for many biomedical applications. Low energy (low power, low intensity (>100kHz, 1 W/cm2) ultrasound is used for monitoring the composition and physico-chemical properties. on the other hand, High energy(high power, high-intensity,≤ 500 kHz ) ultrasound may induce mechanical, physical and chemical / biochemical changes through cavitation that is potentially compromising cell viability. Here in the goal of this work to investigate the potential effect of using therapeutic ultrasound on biological cell viability and integrality. Red blood cells (R.B.Cs) were our choice to investigate cell viability under the therapeutic intensity range of 0.5 – 3 W/cm2). Trypan blue dye exclusion test was to determine the integrity of cellular membrane. Cell membrane osmotic fragility of (R.B.Cs) was measured under series of saline solution osmotic pressure. The electrical signature of (R.B.Cs) was correlated before and after ultrasound irradiation. The results showed no significant alteration in (R.B.Cs) integrity and electrical behavior under US irradiation intensity range. As results using of therapeutic ultrasound intensity range of 0.5 – 3 W/cm2), showed no significant harmful effect on R.B.CS integrity and viability under current experimental conditions.

Comparing the chondrogenic potential of rabbit mesenchymal stem cells derived from the infrapatellar fat pad, periosteum & bone marrow.

Background & objectives:Rabbit model is commonly used to demonstrate the proof of concept in cartilage tissue engineering. However, limited studies have attempted to find an ideal source of rabbit mesenchymal stem cells (MSCs) for cartilage repair. This study aimed to compare the in vitro chondrogenic potential of rabbit MSCs isolated from three sources namely infrapatellar fat pad (IFP), periosteum (P) and bone marrow (BM).Methods:Rabbit MSCs from three sources were isolated and characterized using flow cytometry and multi-lineage differentiation assay. Cell proliferation was assessed using trypan blue dye exclusion test; in vitro chondrogenic potential was evaluated by histology and gene expression and the outcomes were compared amongst the three MSC sources.Results:MSCs from three sources shared similar morphology and expressed >99 per cent positive for CD44 and CD81 and <3 per cent positive for negative markers CD34, CD90 and human leukocyte antigen – DR isotype (HLA-DR). The BM-MSCs and IFP-MSCs showed significantly higher cell proliferation (P<0.001) than the P-MSCs from passage 4. Histologically, BM-MSCs formed a thicker cartilage pellet (P<0.01) with abundant matrix deposition than IFP and P-MSCs during chondrogenic differentiation. The collagen type 2 staining was significantly (P<0.05) higher in BM-MSCs than the other two sources. These outcomes were further confirmed by gene expression, where the BM-MSCs demonstrated significantly higher expression (P<0.01) of cartilage-specific markers (COL2A1, SOX9 and ACAN) with less hypertrophy.Interpretation & conclusions:This study demonstrated that BM-MSCs had superior chondrogenic potential and generated better cartilage than IFP and P-MSCs in rabbits. Thus, BM-MSCs remain a promising candidate for rabbit articular cartilage regeneration.

High-Yield Purification of Giardia intestinalis Cysts from Fecal Samples.

Giardia is an enteric protozoan parasite that causes gastroenteritis in all classes of vertebrates. It is ranked among the leading causes of death in children under 5 years of age. Giardiasis affects approximately 280 million people worldwide annually, a situation exacerbated by the low availability of effective treatments and the lack of a vaccine. In addition, the parasite is difficult to manipulate in in vitro environments, which hampers the development of effective disease management strategies. This article highlights the development of a method for the purification of viable Giardia cysts from fecal samples, verified by a trypan blue dye exclusion test. This protocol produces a 10-fold increase in yield over current methods. By combining sucrose flotation with gated filtration, the protocol significantly reduces the amount of debris in the purified cysts suspension. Cyst viability is verified by a trypan blue dye exclusion test. The ability to purify large quantities of Giardia from fecal samples could advance the development of effective treatments to target this worldwide prevalent parasite. © 2020 Wiley Periodicals LLC. Basic Protocol: Purification of Giardia cysts from fecal samples Support Protocol: Cyst viability test.

MiR-192 enhances sensitivity of methotrexate drug to MG-63 osteosarcoma cancer cells

Chemo-resistance remains a considerable obstacle encountered in osteosarcoma (OS) therapy. Evidence has implied that a reduction in the expression of microRNAs (miRs/miRNAs) leads to exacerbated chemo-resistance. Hence, to better understand the role of miR-192 in the pathogenesis of OS during methotrexate (MTX) treatment, we restore miR-192 in the MG-63 cells and investigate the mechanisms, which are associated with MTX-resistance in OS. Exogenetic overexpression of miR-192 was established by transfecting miR-192 mimics into MG-63 cells using Lipofectamine. Trypan blue dye exclusion test was performed to evaluate the proliferation of the MG-63 cells. Chemo-resistance to MTX was determined using the MTT method after 48 h. ELISA cell death assay was performed to evaluate the apoptosis rate. The quantitative RT-PCR (RT-qPCR) was applied to determine the mRNA expression levels before and after the transfection. Our results illustrated that miR-192 is down-regulated in OS tumor cells. Transfection of miR-192 noticeably alleviated the mRNA expression levels of MMP9, c-Myc, K-Ras, CXCR-4, and ADAMTS compared with the control groups (P-values< 0.05). MTX Combination treatment with miR-192 noticeably elevated the cytotoxic effect of MTX and alleviated its IC50 (P < 0.05). Moreover, miR-192 significantly increased the apoptotic effect of MTX. These results implied that miR-192 enhances the sensitivity of MG-63 cells to MTX. Collectively, our results elucidated that miR-192 contributes to chemo-sensitizing MG-63 cells to MTX, and could be considered as a promising agent to overcome MTX-resistance in OS.

Tumor Exosomes Mediate the Horizontal Transfer of DNA Gene Mutation

INTRODUCTIONTumor‐derived exosomes (TEXs) are crucial in shaping the local tumor microenvironment to promote cancer progression by advancing tumor metastasis. Recently, we have demonstrated that the activation of Toll‐like receptor 4 (TLR4) boosts the immunosuppressive ability of exosomes released by tumor cells. In addition to RNA and proteins, TEXs also transport dsDNA that reflects the mutational status of their parental cells. The purpose of this study is to verify whether exosomes shed by TLR4‐activated tumor cells are able to induce a malignant transformation of normal recipient cells through the horizontal transfer of mutated DNA.METHODSSW480 tumor cells were treated with LPS (1μg/ml) and TEXs were isolated from cell supernatants by polymer precipitation‐based method. TP53R273H and KRASG12V mutations have been identify by droplet digital PCR on purified exoDNA. Normal colon epithelial cells (CCD841) were used as recipient cells and internalization of Did‐labelled vesicles was evaluated by immunofluorescence. Vitality/proliferation (Trypan Blue dye exclusion test), migration capacity (Scratch assay) and mutational status (Droplet digital PCR) of CCD841 cells were monitored after the treatment with exosomes.RESULTSSW480‐derived exosomes carried dsDNA fragments in size range of 2–10 kbp containing TP53R273H and KRASG12V mutated sequences. Exosomes were efficiently internalized by CCD841 cells within 6 hours and elicited activation of signaling pathways that induced an increase in cell proliferation and migration capacity. Copies of TP53 R273H mutated DNA and mRNA were found within recipient cells up to one month without exosomal treatment. The activation with LPS stimulated the packaging of mutated DNA into exosomes. Consequently, mutated DNA was found in greater amounts in recipient cell treated with exosomes released by TLR4‐activated cells.CONCLUSIONSThe activation of TLR4 modulates the packaging of mutated genes in exosomes, which are efficiently transferred in normal recipient cells. Although we observed that mutated TP53 gene is transcribed in recipient cells, further studies are needed to assess whether mutated gene are integrated into genome.Support or Funding InformationThis project was supported by Grant Interreg ARTE (J22F170001005)

Potent Suppression of Proliferation of Breast Carcinoma Cells by a Novel Anthranilic Acid Derivative

Background/objective: Chemotherapy remains the mainstay therapy for the most intrusive-type of breast carcinoma, triple-negative breast cancer (TNBC) that has a higher tendency for visceral metastases, relapses, and poor prognoses. It is well recognized that the reactivation of a ribonucleoprotein enzyme, telomerase is among the key determinants of breast carcinogenesis, cellular immortalization and metastatic progression. Therefore, our objectives were to assess (i) short- and (ii) long-term effects of a novel anthranilic acid (GV6) developed at our institute and compare it to a known analogue, BIBR1532 on TNBC (MDA-MB 231) and non-TNBC (MCF-7) cells. Methods: Seeded TNBC and non-TNBC flasks were supplemented with 25 µM of either BIBR 1532 or GV6 or solvent (DMSO) alone for 14 (Short-term) or 27 (Long-term) days. Trypan-blue dye exclusion test was utilized to determine viable cells, senescence-associated β-galactosidase activity was employed to detect senescent cells and qPCR was used to quantitate transcript abundance. Results: Cell viability assay revealed that short- and long-term growth inhibitions of TNBC and non-TNBC cells were comparable between BIBR1532 and GV6. Cytochemical detection of βGalactosidase demonstrated that GV6 was equally effective in inducing replicative senescence in treated TNBC and non-TNBC cells. Short- and long-term regimen of BIBR1532 and GV6 showed similar drug-induced downregulation of hTERT in TNBC and non-TNBC cells. Conclusion: Results indicate that GV6 is an equally potent inhibitor of hTERT that induces growth impedance and triggers senescence in TNBC, as well as non-TNBC cells and merits further studies for improved treatment options.

Comparative Analysis of Two Automated Fat-processing Systems.

Background:Plastic surgeons desire more efficient methods of processing lipoaspirate when performing fat grafting procedures. We compared, in a preclinical study, the quantity and quality of lipoaspirate processed by a novel Poloxamer Wash, Absorption, mesh filtration System (PWAS) to a frequently used Ringer’s Lactate wash, Decant, and mesh filtration System (RLDS).Methods:Lipoaspirate from 10 patients was processed with the RLDS and PWAS systems. The processed lipoaspirate from each device was centrifuged to quantify the amount of fat, free oil, and aqueous components remaining in the fat graft. A trypan blue dye exclusion test assessed cell viability. The processing time for the lipoaspirate was also measured.Results:The 10-patient average fat volume processed and available for grafting was similar using both systems. The adipose volume fraction of PWAS was greater (89% ± 3%) than RLDS (76% ± 10%, P = 0.02). The trypan blue exclusion values and processing time were similar for both systems. Oil was efficiently removed from the lipoaspirate, and both systems processed fat efficiently.Conclusion:The PWAS effectively cleans lipoaspirate with increased fat concentration.