Culturing and Scaling up Stem Cells of Dental Pulp Origin Using Microcarriers

Ectomesenchymal stem cells derived from the dental pulp are of neural crest origin, and as such are promising sources for cell therapy and tissue engineering. For safe upscaling of these cells, microcarrier-based culturing under dynamic conditions is a promising technology. We tested the suitability of two microcarriers, non-porous Cytodex 1 and porous Cytopore 2, for culturing well characterized dental pulp stem cells (DPSCs) using a shake flask system. Human DPSCs were cultured on these microcarriers in 96-well plates, and further expanded in shake flasks for upscaling experiments. Cell viability was measured using the alamarBlue assay, while cell morphology was observed by conventional and two-photon microscopies. Glucose consumption of cells was detected by the glucose oxidase/Clark-electrode method. DPSCs adhered to and grew well on both microcarrier surfaces and were also found in the pores of the Cytopore 2. Cells grown in tissue culture plates (static, non-shaking conditions) yielded 7 × 105 cells/well. In shake flasks, static preincubation promoted cell adhesion to the microcarriers. Under dynamic culture conditions (shaking) 3 × 107 cells were obtained in shake flasks. The DPSCs exhausted their glucose supply from the medium by day seven even with partial batch-feeding. In conclusion, both non-porous and porous microcarriers are suitable for upscaling ectomesenchymal DPSCs under dynamic culture conditions.

Synthesis and Antiparasitic Activity of New Conjugates—Organic Drugs Tethered to Trithiolato-Bridged Dinuclear Ruthenium(II)–Arene Complexes

Tethering known drugs to a metalorganic moiety is an efficient approach for modulating the anticancer, antibacterial, and antiparasitic activity of organometallic complexes. This study focused on the synthesis and evaluation of new dinuclear ruthenium(II)–arene compounds linked to several antimicrobial compounds such as dapsone, sulfamethoxazole, sulfadiazine, sulfadoxine, triclosan, metronidazole, ciprofloxacin, as well as menadione (a 1,4-naphtoquinone derivative). In a primary screen, 30 compounds (17 hybrid molecules, diruthenium intermediates, and antimicrobials) were assessed for in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in noninfected host cells (human foreskin fibroblasts, HFF) was determined by an alamarBlue assay. When assessed at 1 µM, five compounds strongly impaired parasite proliferation by >90%, and HFF viability was retained at 50% or more, and they were further subjected to T. gondii β-gal dose-response studies. Two compounds, notably 11 and 13, amide and ester conjugates with sulfadoxine and metronidazole, exhibited low IC50 (half-maximal inhibitory concentration) values 0.063 and 0.152 µM, and low or intermediate impairment of HFF viability at 2.5 µM (83 and 64%). The nature of the anchored drug as well as that of the linking unit impacted the biological activity.

3D nyomtatható MED610 polimerből kioldódó monomerek kvalitatív analízise és ezek hatása sejtekre

A háromdimenziós (3D) nyomtatást, dinamikusan fejlődő technikájának köszönhetően, az ipari területek után az orvostudomány is egyre szélesebb körben alkalmazza. A fogászatban elterjedt 3D nyomtatáshoz használt polimerek orális környezetben való használata során kulcskérdés a biokompatibilitás. Célkitűzésünk a MED610 (Stratasys, USA) polimerből készített mintákból kioldódó komponensek azonosítása, valamint ezek hatásának megfigyelése csont eredetű sejtek osztódására. A kutatás során különböző méretű és felületnagyságú objektumokat nyomtattunk Objet 30 OrthoDesk (Stratasys, USA) nyomtatóval, és felületkezeltük a gyártó utasításai szerint. Ezen minták közvetve vagy közvetlenül érintkeztek Sarcoma Osteogenic-2 (SAOS-2) sejtekkel. A MED610 kioldódási vizsgálatát metanolban végeztük, amit gázkromatográfia–tömegspektrometria kapcsolt módszerrel (GC-MS) analizáltunk. A proliferációra gyakorolt hatásokat alamarBlue assay segítségével mértük. A vizsgálatok alapján izobornil-akrilát, hidroxiciklohexil-fenil-keton és kaprolakton oldódik ki nagyobb mennyiségben a MED610 polimerből. A sejtéletképességi vizsgálatok eredményei alapján pedig elmondható, hogy a MED610 a felületnöveléssel párhuzamosan szignifikánsan csökkenti a SAOS-2 sejtek proliferációját, amelyet az anyaggal történő közvetlen érintkezés felerősít. Az általunk megfigyelt hatást valószínűleg a MED610-ből kioldódó toxikus anyagok okozzák, amelyek a nagyobb felület következtében gyorsabban oldódnak ki, ezáltal nagyobb koncentrációban találhatóak meg a sejtek környezetében.

Docking and antibacterial activity of novel nontoxic 5-arylidenepyrimidine-triones as inhibitors of NDM-1 and MetAP-1

Background: Antibiotic resistance, which occurs through the action of metallo-β-lactamases (NDM-1), is a serious problem in the treatment of infectious diseases. Therefore, the discovery of new NDM-1 inhibitors and promising antibacterial agents as inhibitors of alternative targets (MetAP-1) is important. Method & results: In this study, a virtual library of 5-arylidene barbituric acids was created and molecular docking was performed for identification of novel possible inhibitors of NDM-1 and MetAP-1. Antibacterial activity (agar well-diffusion assay) and cytotoxicity (alamarBlue assay) of perspective compounds were evaluated. Pharmacokinetic profiles and molecular properties were predicted. Conclusion: We have identified possible novel inhibitors of NDM-1 and MetAP-1 with bacteriostatic activity, most of which are not cytotoxic and have potential excellent drug-likeness properties.

Triptolide Modulates the Expression of Inflammation-Associated lncRNA-PACER and lincRNA-p21 in Mycobacterium tuberculosis–Infected Monocyte-Derived Macrophages

Triptolide is a diterpene triepoxide, which performs its biological activities via mechanisms including induction of apoptosis, targeting of pro-inflammatory cytokines, and reshaping of the epigenetic landscape of target cells. However, the targeting of long non-coding RNAs (lncRNAs) by triptolide has not yet been investigated, despite their emerging roles as key epigenetic regulators of inflammation and immune cell function during Mycobacterium tuberculosis (Mtb) infection. Hence, we investigated whether triptolide targets inflammation-associated lncRNA-PACER and lincRNA-p21 and how this targeting associates with Mtb killing within monocyte-derived macrophages (MDMs).Using RT-qPCR, we found that triptolide induced the expression of lincRNA-p21 but inhibited the expression of lncRNA-PACER in resting MDMs in a dose- and time-dependent manner. Moreover, Mtb infection induced the expression of lincRNA-p21 and lncRNA-PACER, and exposure to triptolide before or after Mtb infection led to further increase of Mtb-induced expression of these lncRNAs in MDMs. We further found that contrary to lncRNA-PACER, triptolide time- and dose-dependently upregulated Ptgs-2, which is a proximal gene regulated by lncRNA-PACER. Also, low-concentration triptolide inhibited the expression of cytokine IL-6, a known target of lincRNA-p21. Mtb infection induced the expression of IL-6 and Ptgs-2, and triptolide treatment further increased IL-6 but decreased Ptgs-2 expression in Mtb-infected MDMs. The inverse relation between the expression of these lncRNAs and their target genes is concordant with the conception that these lncRNAs mediate, at least partially, the cytotoxic and/or anti-inflammatory activities of triptolide in both resting and activated MDMs. Using the CFU count method, we found that triptolide decreased the intracellular growth of Mtb HN878. The alamarBlue assay showed that this decreased Mtb HN878 growth was not as a result of direct targeting of Mtb HN878 by triptolide, but rather evoking MDMs’ intracellular killing mechanisms which we speculate could include triptolide-induced enhancement of MDMs’ effector killing functions mediated by lncRNA-PACER and lincRNA-p21. Altogether, these results provide proof of the modulation of lncRNA-PACER and lincRNA-p21 expression by triptolide, and a possible link between these lncRNAs, the enhancement of MDMs’ effector killing functions and the intracellular Mtb-killing activities of triptolide. These findings prompt for further investigation of the precise contribution of these lncRNAs to triptolide-induced activities in MDMs.

Anti-Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer Studies on Clausena excavata Burum. f. (Rutaceae) Leaves

Background. Clausena excavata Burum. f. has long been applied in ethnomedicine for the treatment of various disorders like rhinitis, headache, cough, wound healing, fever, and detoxification. This study is aimed at investigating the antibacterial activity against Enterococcus faecalis ATCC 49532 using AlamarBlue assay and atomic force microscopy (AFM) as well as the cytotoxicity, anticancer, and phytotoxicity of C. excavata. Method. Bacterial cell viability was performed by using microplate AlamarBlue assay. Atomic force microscopy was used to determine morphological changes in the surface of bacterial cells. Cytotoxicity and phytotoxicity were determined by brine shrimp lethality and Lemna minor bioassay. Caco-2 (colorectal adenocarcinoma) cell line was used for the evaluation of the anticancer effects. Result. Among the fractions tested, ethyl acetate (EA) fraction was found to be active with minimum inhibitory concentration (MIC) of 750 μg/mL against E. faecalis, but other fractions were found to be insensitive to bacterial growth. Microscopically, the EA fraction-treated bacteria showed highly damaged cells with their cytoplasmic content scattered all over. The L C 50 value of the EA fraction against brine shrimp was more than 1000 μg/mL showing the nontoxic nature of this fraction. Chloroform (CH), EA, and methanol (MOH) fractions of C. excavata were highly herbicidal at the concentration of 1000 μg/mL. EA inhibited Caco-2 cell line with an I C 50 of 20 μg/mL. Conclusions. This study is the first to reveal anti-E. faecalis property of EA fraction of C. excavata leaves, natural herbicidal, and anticancer agents thus highlight the potential compound present in its leaf which needs to be isolated and tested against multidrug-resistant E. faecalis.

Xao tam phan (Paramignya trimera) methanol extract induced apoptosis in hepatocellular carcinoma HepG2 cell line in vitro

Introduction: Xao Tam Phan (Paramignya trimera) has long been used in Viet Nam as an herbal medicine for the treatment of Hepatitis, hepatocellular carcinoma, and diabetes. This study aimed to determine the anti-proliferation effect of Paramignya trimera extract (P. trimera extract) on HepG2 hepatocellular carcinoma cells. Methods: AlamarBlue assay was used to determine the IC50 values of P. trimera extract on HepG2 cells. Adipose-derived stem cells (ADSCs) was used as normal cell control. For apoptosis examination, P. trimera extract-treated HepG2 cells were incubated with Annexin V/Propidium iodide (PI). Then they have been analyzed their expression of Annexin-V and PI by flow cytometry. The cell nuclear degradation also was evaluated by PI/Hoechst 33342 staining assay. Results: Doxorubicin and P. trimera extract IC50 values on HepG2 cells were 55.13 +/- 2.028 ng/ml and 582.533 +/- 16.521 mg/ml, respectively. Those on ADSCs were 5.96 +/- 0.56 ng/ml and 268.976 +/- 19.325 mg/ml, respectively. Side effect index value (SEI) of P. trimera extract was 2.175 +/- 0.12, and the SEI of doxorubicin was 8.71 +/- 0.36. Flow cytometry analysis indicated significant apoptosis on P. trimera extract-treated HepG2 cells at a dose of 500 mg/ml (32.39 +/- 2.28% apoptotic cells, and 14.63 +/- 1.59% necrotic cells). Nuclear aggregation and degradation was seen on 500 mg/ml P. trimera treated HepG2 cells. Conclusion: P. trimera extract could inhibit HepG2 hepatocellular carcinoma cell proliferation by inducing apoptosis.

Pyomelanin Secretion in Madurella mycetomatis Interferes with Spectrophotometric Endpoint Reading Using the Sensititre YeastOne alamarBlue Assay but Not with Visual Endpoint Reading

ABSTRACT The use of the Sensititre YeastOne YO10 alamarBlue assay for the in vitro susceptibility testing of Madurella mycetomatis was evaluated in M. mycetomatis isolates with and without pyomelanin secretion. Pyomelanin secretion did not influence visual endpoint reading; however, it caused a shift in peak absorbance from 570 nm to 620 nm when read spectrophotometrically. Therefore, when choosing the method for endpoint reading, the presence of pyomelanin should be considered.

Direct-Contact Cytotoxicity Evaluation of CoCrFeNi-Based Multi-Principal Element Alloys

Transition metal multi-principal element alloys (MPEAs) are novel alloys that may offer enhanced surface and mechanical properties compared with commercial metallic alloys. However, their biocompatibility has not been investigated. In this study, three CoCrFeNi-based MPEAs were fabricated, and the in vitro cytotoxicity was evaluated in direct contact with fibroblasts for 168 h. The cell viability and cell number were assessed at 24, 96, and 168 h using LIVE/DEAD assay and alamarBlue assay, respectively. All MPEA sample wells had a high percentage of viable cells at each time point. The two quaternary MPEAs demonstrated a similar cell response to stainless steel control with the alamarBlue assay, while the quinary MPEA with Mn had a lower cell number after 168 h. Fibroblasts cultured with the MPEA samples demonstrated a consistent elongated morphology, while those cultured with the Ni control samples demonstrated changes in cell morphology after 24 h. No significant surface corrosion was observed on the MPEAs or stainless steel samples following the cell culture, while the Ni control samples had extensive corrosion. The cell growth and viability results demonstrate the cytocompatibility of the MPEAs. The biocompatibility of MPEAs should be investigated further to determine if MPEAs may be utilized in orthopedic implants and other biomedical applications.