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Almost every death in young patients with an advanced skin tumor is caused by melanoma. Today, with the help of modern treatments, these patients survive longer or can even achieve a cure. Advanced stage melanoma is frequently related with poor prognosis and physicians still find this disease difficult to manage due to the absence of a lasting response to initial treatment regimens and the lack of randomized clinical trials in post immunotherapy/targeted molecular therapy settings. New therapeutic targets are emerging from preclinical data on the genetic profile of melanocytes and from the identification of molecular factors involved in the pathogenesis of malignant transformation. In the current paper, we present the diagnostic challenges, molecular biology and genetics of malignant melanoma, as well as the current therapeutic options for patients with this diagnosis.
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Injectable platelet-rich fibrin (iPRF) is a frequently used platelet concentrate used for various medical purposes both in veterinary and human medicine due to the regenerative potential of hard and soft tissues, and also because of its antimicrobial effectiveness. This in vitro study was carried out to assess the cumulative antimicrobial and antibiofilm effect of iPRF functionalized with a multifunctional glycoprotein, human lactoferrin (Lf). Thus, the ability to potentiate cell proliferation was tested on keratinocytes and evaluated by the CCK8 test. The combinations of iPRF and Lf induced an increase in the proliferation rate after 24 h. The average cell viability of treated cultures (all nine variants) was 102.87% ± 1.00, and the growth tendency was maintained even at 48 h. The highest proliferation rate was observed in cultures treated with 7% iPRF in combination with 50 µg/mL of Lf, with an average viability of 102.40% ± 0.80. The antibacterial and antibiofilm activity of iPRF, of human lactoferrin and their combination were tested by agar-well diffusion (Kirby-Bauer assay), broth microdilution, and crystal violet assay against five reference bacterial strains. iPRF showed antimicrobial and antibiofilm potential, but with variations depending on the tested bacterial strain. The global analysis of the results indicates an increased antimicrobial potential at the highest concentration of Lf mixed with iPRF. The study findings confirmed the hypothesized enhanced bioactive properties of functionalized iPRF against both Gram-positive and Gram-negative biofilm-producing bacteria. These findings could be further applied, but additional studies are needed to evaluate the mechanisms that are involved in these specific bioactive properties.
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Anti-Infecciosos , Plasma Rico em Plaquetas , Humanos , Lactoferrina/farmacologia , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Bactérias , Bactérias Gram-NegativasRESUMO
The aim of the present study was to correlate the antioxidant, antimicrobial, and cytotoxic activities of hydroalcoholic extracts obtained from the aerial parts of three Dracocephalum moldavica L. cultivars with their polyphenolic compositions. The polyphenols were identified and quantified using spectrophotometrical methods and LC-MS analysis. Their antioxidant capacities were assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. Their in vitro antimicrobial efficacies were assessed using the agar well diffusion and broth microdilution methods. Their cytotoxicity was investigated on normal diploid foreskin fibroblasts (BJ) and on colorectal adenocarcinoma (DLD-1) cell lines. The results pointed out significant amounts of polyphenolic compounds in the compositions of the tested cultivars, with rosmarinic acid as the main compound (amounts ranging between 5.337 ± 0.0411 and 6.320 ± 0.0535 mg/mL). All three cultivars displayed significant antioxidant (IC50 ranging between 35.542 ± 0.043 and 40.901 ± 0.161 µg/mL for the DPPH assay, and for the FRAP assay 293.194 ± 0.213 and 330.165 ± 0.754 µmol Trolox equivalent/mg dry vegetal material) and antimicrobial potential (especially towards the Gram-positive bacteria), as well as a selective toxicity towards the tumoral line. A significant positive correlation was found between antioxidant activity and the total phenolic acids (r2 = 0.987) and polyphenols (r2 = 0.951). These findings bring further arguments for strongly considering D. moldavica cultivars as promising vegetal products, which warrants further investigation.
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Anti-Infecciosos , Antineoplásicos , Antioxidantes/química , Extratos Vegetais/química , Polifenóis/farmacologia , Anti-Infecciosos/farmacologiaRESUMO
In the presented study, the effects of ROCK inhibitor Y-27632, antifreeze protein III, and boron at two different doses were investigated on the spermatological parameters of Ankara buck semen after freeze−thawing. Ejaculates were collected from bucks using an electroejaculator during the breeding season. The ejaculates that showed appropriate characteristics were pooled and used in the dilution and freezing of semen. The extender groups were formed by adding two different doses of three different additives (ROCK inhibitor Y-27632, 5 and 20 µM; antifreeze protein III, 1 and 4 µg/mL; boron, 0.25 and 1 mM) to the control extender. The semen was diluted with the different extenders at 35−37 °C and loaded into straws. Sperm samples frozen in liquid nitrogen vapors, following equilibration, were stored in liquid nitrogen. It was observed that extender supplementation improved post-thaw motility of Ankara buck semen after freeze−thawing. Differences were significant (p < 0.01) for 5 and 10 µM doses of ROCK inhibitor (71.82% and 74.04 % motility), as well as for 0.25 and 1 mM doses of boron (76.36% and 72.08% motility), compared to the control group (66.15% motility). With respect to the evaluation of acrosomal integrity and mitochondrial activity after freeze−thawing, although supplementation provided protection at all doses, the efficacy was not statistically significant (p > 0.05). It was observed that DNA damage was improved by antifreeze protein III at 1 µg/mL (1.23% ± 0.23%) and by boron at all doses (0.25 mM: 1.83% and 1 mM: 1.18%) compared to the control group (3.37%) (p < 0.01), following the thawing process. In the present study, it was determined that some additives added to the extender provided significant improvements in buck spermatozoa motility and DNA damage after thawing.
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Preservação do Sêmen , Sêmen , Masculino , Humanos , Preservação do Sêmen/métodos , Boro/farmacologia , Boro/metabolismo , Quinases Associadas a rho/metabolismo , Criopreservação/métodos , Crioprotetores/farmacologia , Proteínas Anticongelantes/metabolismo , Nitrogênio/metabolismoRESUMO
The concern for implementing bioactive nutraceuticals in antioxidant-related therapies is of great importance for skin homeostasis in benign or malignant diseases. In order to elucidate some novel insights of Lycium barbarum (Goji berry) activity on skin cells, the present study focused on its active compound zeaxanthin. By targeting the stemness markers CD44 and CD105, with deep implications in skin oxidative stress mechanisms, we revealed, for the first time, selectivity in zeaxanthin activity. When applied in vitro on BJ human fibroblast cell line versus the A375 malignant melanoma cells, despite the moderate cytotoxicity, the zeaxanthin-rich extracts 1 and 2 were able to downregulate significantly the CD44 and CD105 membrane expression and extracellular secretion in A375, and to upregulate them in BJ cells. At mechanistic level, the present study is the first to demonstrate that the zeaxanthin-rich Goji extracts are able to influence selectively the mitogen-activated protein kinases (MAPK): ERK, JNK and p38 in normal BJ versus tumor-derived A375 skin cells. These results point out towards the applications of zeaxanthin from L. barbarum as a cytoprotective agent in normal skin and raises questions about its use as an antitumor prodrug alone or in combination with standard therapy.
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Adesão Celular/efeitos dos fármacos , Lycium/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Extratos Vegetais/farmacologia , Zeaxantinas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Frutas/química , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Extratos Vegetais/isolamento & purificação , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo , Zeaxantinas/isolamento & purificaçãoRESUMO
Background and objective: The aim of the present study was to establish a new differentiation protocol using cannabidiol (CBD) and vitamin D3 (Vit. D3) for a better and faster osteogenic differentiation of dental tissue derived mesenchymal stem cells (MSCs). Materials and methods: MSCs were harvested from dental follicle (DFSCs), dental pulp (DPSCs), and apical papilla (APSCs) of an impacted third molar of a 17-year old patient. The stem cells were isolated and characterized using flow cytometry; reverse transcription polymerase chain reaction (RT-PCR); and osteogenic, chondrogenic, and adipogenic differentiation. The effects of CBD and Vit. D3 on osteogenic differentiation of dental-derived stem cell were evaluated in terms of viability/metabolic activity by alamar test, expression of collagen1A, osteopontin (OP), osteocalcin (OC), and osteonectin genes and by quantification of calcium deposits by alizarin red assay. Results: Stem cell characterization revealed more typical stemness characteristics for DFSCs and DPSCs and atypical morphology and markers expression for APSCs, a phenotype that was confirmed by differences in multipotential ability. The RT-PCR quantification of bone matrix proteins expression revealed a different behavior for each cell type, APSCs having the best response for CBD. DPSCs showed the best osteogenic potential when treated with Vit. D3. Cultivation of DFSC in standard stem cell conditions induced the highest expression of osteogenic genes, suggesting the spontaneous differentiation capacity of these cells. Regarding mineralization, alizarin red assay indicated that DFSCs and APSCs were the most responsive to low doses of CBD and Vit. D3. DPSCs had the lowest mineralization levels, with a slightly better response to Vit. D3. Conclusions: This study provides evidence that DFSCs, DPSCs, and APSCs respond differently to osteoinduction stimuli and that CBD and Vit. D3 can enhance osteogenic differentiation of these types of cells under certain conditions and doses.
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Canabidiol , Células-Tronco Mesenquimais , Adolescente , Canabidiol/farmacologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colecalciferol/farmacologia , Humanos , OsteogêneseRESUMO
OBJECTIVES: Tooth bleaching is one of the most required dental esthetic treatments. However, it can generate side effects like oral irritation, enamel alteration, tooth sensitivity, especially caused by hydrogen peroxide, the main bleaching component of the commercial products. Therefore, development of new tooth bleaching agents, based on natural products, with comparable esthetic results and lower side effects is needed. The aim of this study was to evaluate the biological effects and bleaching efficacy of four experimental bleaching agents, derived from fruit juices, against the commercially available Opalescence (Ultradent, USA). MATERIALS AND METHODS: Organic acid composition of the gels was characterized by HPLC. Bleaching efficiency was tested by spectrophotometry on composite restorative materials. Biological testing was done in vitro, on human fibroblasts. Cells were exposed to dilutions of the bleaching gel-conditioned medium. Viability was measured by MTS, apoptosis by FACS-AnnexinV FITC/Propidium iodide, NF-kB activation by western blot, malondyaldehide, and superoxide dismutase activity by spectrophotometry. RESULTS: All gels exhibited physical stability and dental bleaching capabilities. Experimental gels induced significantly better viability and apoptosis rates, lower lipid peroxidation, and increased antioxidant defense, compared to Opalescence. CONCLUSIONS: The studied experimental gel formulations exhibited a good safety profile in vitro, as well as bleaching efficiency on restorative composite materials. CLINICAL RELEVANCE: These data open new possibilities for the use of new natural products in dental bleaching treatments that can insure significant esthetic results and lower side effects.
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Extratos Vegetais/farmacologia , Clareadores Dentários/farmacologia , Antioxidantes/análise , Apoptose/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Resinas Compostas/química , Combinação de Medicamentos , Fibroblastos/efeitos dos fármacos , Sucos de Frutas e Vegetais/toxicidade , Géis , Técnicas In Vitro , Peroxidação de Lipídeos , Peróxidos , Extratos Vegetais/toxicidade , Polivinil , Espectrofotometria , Clareadores Dentários/toxicidade , Ureia/análogos & derivadosRESUMO
New Pd(II) complexes of 1,7-bis(2-methoxyphenyl)hepta-1,6-diene-3,5-dione were synthesized and structurally characterized. The complexes were tested in vitro on human colon and hepatic carcinoma cell lines, normal hepatic cells and hematopoietic progenitor cells. Biological tests proved that Pd(II) complexes 1 and 2 (containing a curcumin derivative) exhibit a strong in vitro antitumor effect against the cells derived from human colorectal carcinoma and the hepatic metastasis of a colorectal carcinoma. Complex 1 has an outstanding inhibitory effect against BRAF-mutant colon carcinoma and hepatocarcinoma cell growth; 1 and 2 are both more active than the free ligand and have the capacity to trigger early apoptotic processes. By flow cytometric measurements, an important decrease of prominin-1 (CD133) molecule expression on tumor cells membrane was identified in cell populations subjected to 1 and 2. Quantitative immune enzymatic assay proved restrictions in stem cell factor (SCF) release by treated tumor cells. Although less cytotoxic, the free ligand inhibits the surface marker CD133 expression in hepatocarcinoma cells, and in HT-29 colon carcinoma. The new synthesized Pd(II) complexes 1 and 2 exhibit an important potential through their selective cytotoxic activity and by targeting the stem-like tumor cell populations, which leads to the tumor growth arrest and prevention of metastasis.
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Antígeno AC133/metabolismo , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Paládio/química , Fator de Células-Tronco/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Neoplasias Hepáticas , Compostos Organometálicos/químicaRESUMO
The aim of the present study was to isolate human mesenchymal stem cells (MSCs) from palatal connective and periodontal granulation tissues and to comparatively evaluate their properties. MSCs were isolated using the explant culture method. Adherence to plastic, specific antigen makeup, multipotent differentiation potential, functionality, and ultrastructural characteristics were investigated. The frequency of colony-forming unit fibroblasts for palatal-derived mesenchymal stem cells (pMSCs) was significantly higher than that of granulation tissue-derived mesenchymal stem cells (gtMSCs). A significantly higher population doubling time and lower migration potential were recorded for gtMSCs than for pMSCs. Both cell lines were positive for CD105, CD73, CD90, CD44, and CD49f, and negative for CD34, CD45, and HLA-DR, but the level of expression was different. MSCs from both sources were relatively uniform in their ultrastructure. Generally, both cell lines possessed a large, irregular-shaped euchromatic nucleus, and cytoplasm rich in mitochondria, lysosomes, and endoplasmic reticulum. The periphery of the plasma membrane displayed many small filopodia. MSCs from both cell lines were successfully differentiated into osteogenic, adiopogenic, and chondrogenic lineages. Both healthy and diseased tissues may be considered as valuable sources of MSCs for regenerative medicine owing to the high acceptance and fewer complications during harvesting.
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Tecido Conjuntivo , Células-Tronco Mesenquimais/fisiologia , Mucosa Bucal/citologia , Adulto , Antígenos CD/análise , Diferenciação Celular , Membrana Celular/ultraestrutura , Movimento Celular , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Perfilação da Expressão Gênica , Antígenos HLA-DR/análise , Voluntários Saudáveis , Humanos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/ultraestrutura , Microscopia , Organelas/ultraestrutura , Doenças Periodontais , Adulto JovemRESUMO
A series of alkenyl-substituted titanocene compounds have been supported on the mesoporous silica-based material KIT-6. The corresponding functionalised materials were completely characterised by different techniques (solid-state multinuclear NMR spectroscopy, IR spectroscopy, N2 adsorption-desorption isotherms, X-ray fluorescence and diffraction, SEM and TEM) to observe the incorporation of the titanocene derivatives on the external surface of the material KIT-6. Both the titanocene compounds and the materials were tested in vitro against a wide variety of human cancer and normal cell lines. A very high cytotoxicity of the synthesised titanocene derivatives (IC50 values in the range of those described in the literature for the most active cytotoxic titanocene compounds), with selectivity towards cancer cell lines was observed. The cytotoxic activity of the materials is the highest reported to date for titanocene-functionalised materials. In addition, higher Ti uptake (from 4 to 23% of the initial amount of Ti) of the cells treated with materials was observed with respect to those treated with "free" titanocene derivatives (which gave Ti uptake values from 0.4 to 4.6% of the initial amount of Ti). Additional experiments with the titanocene derivatives and the functionalised materials revealed that changes to the morphological and functional dynamics of apoptosis occurred when the active titanocene species were incorporated into mesoporous materials. In addition, the materials could induce programmed cell death in tumour cell populations by impairing the damaged DNA repair mechanisms and by upregulation of intrinsic and extrinsic apoptotic signalling pathways.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Nanoestruturas/química , Compostos Organometálicos/química , Antineoplásicos/síntese química , Linhagem Celular , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Células HEK293 , Humanos , Células MCF-7 , Conformação Molecular , Nanoestruturas/ultraestrutura , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Porosidade , Dióxido de Silício/química , Proteína bcl-X/metabolismoRESUMO
PURPOSE: Glioblastoma stem cells (GSCs), responsible for the dismal disease prognosis after conventional treatments, are driven by overactive signaling pathways, such as PI3K/ AKT/mTOR and RAS/RAF/MAPK. The objective of our study was to target in vitro-GSCs by combining metformin (Met) as a mTOR inhibitor, with sorafenib (Soraf) as a RAF inhibitor. METHODS: GSCs cultured under basal conditions were treated with Met, temozolomide (TMZ), Soraf, Met+TMZ and Met+Soraf; as untreated arm served as control. At 4 hrs of drug exposure, we measured the level of reactive oxygen species (ROS) by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, apoptosis by prodium iodide (PI)-V Annexin staining and efflux pump activity by using the fluorescent dye rhodamine 123. At 24 hrs, we measured cell proliferation by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and malondialdehyde (MDA) levels. MTT results were compared with corresponding measurements on cultures of non-stem glioblastoma cells and osteoblasts. RESULTS: Met+Soraf exerted the highest antiproliferative effects in GSCs and non-stem glioblastoma cells (p<0.001). Both Met and Soraf monotherapy exhibited a selective cytotoxic effect on GSCs (p<0.001), while no effect was detected on non-stem glioblastoma cells (p>0.05). Soraf, but not Met, impacted the proliferation of normal cells. Soraf displayed synergism with Met in producing high levels of ROS, decreasing efflux pump activity and generating the highest apoptotic rates when compared to either drug alone (p<0.001). CONCLUSION: GSCs were highly sensitive to the combination of Met and Soraf which reduced cell proliferation, increased oxidative stress, inhibited efflux pump activity and ultimately killed GSCs. We strongly believe that these results warrant further in vivo exploration.
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Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Metformina/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Niacinamida/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinases raf/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Humanos , Peroxidação de Lipídeos , Células-Tronco Neoplásicas/metabolismo , Niacinamida/administração & dosagem , Estresse Oxidativo , Rodamina 123/metabolismo , Sorafenibe , TemozolomidaRESUMO
Considering that Sorbus aucuparia fruits have been underutilized despite their tremendous potential, this study aimed to correlate the in vitro antioxidant, antibacterial and cell-protective abilities of fruit extracts derived from Sorbus aucuparia Romanian cultivars with their phytochemical composition. Therefore, following the preparation of ethanolic and carotenoid extracts, phytochemical screening was performed using UV-Vis and HPLC-DAD-ESI-MS methods. The antioxidant activity was analyzed using DPPH and FRAP tests. As the results revealed high contents of bioactive compounds (polyphenols 1.11 mg GAE/g DM, flavonoids 430.06 µg QE/g DM and carotenoids 95.68 µg/g DM) and an important antiradical action (DPPH 24.51 mg/mL and FRAP 0.016 µM TE/mL), we chose to further examine the fruits' biological properties. The antibacterial capacity was assessed employing agar well diffusion and broth microdilution techniques, with fruits displaying an intense activity against MSSA, MRSA and Enterococcus faecalis, but also E. coli and Pseudomonas aeruginosa. The cell-protective activity was analyzed on gentamicin-stressed renal cells, through MTT and Annexin V-FITC assays. Importantly, a significant increase in viability was registered on stressed cells following extract administration in low doses; nevertheless, viability was noticed to decline when exposed to elevated concentrations, potentially due to the cumulative actions of the extract and gentamicin. These findings offer novel light on the antibacterial activity of Sorbus aucuparia Romanian cultivars, as well as their cell-protective ability in renal cell injury.
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Despite the numerous advantages of allogeneic hematopoietic stem cell transplants (allo-HSCT), there exists a notable association with risks, particularly during the preconditioning period and predominantly post-intervention, exemplified by the occurrence of graft-versus-host disease (GVHD). Risk stratification prior to symptom manifestation, along with precise diagnosis and prognosis, relies heavily on clinical features. A critical imperative is the development of tools capable of early identification and effective management of patients undergoing allo-HSCT. A promising avenue in this pursuit is the utilization of proteomics-based biomarkers obtained from non-invasive biospecimens. This review comprehensively outlines the application of proteomics and proteomics-based biomarkers in GVHD patients. It delves into both single protein markers and protein panels, offering insights into their relevance in acute and chronic GVHD. Furthermore, the review provides a detailed examination of the site-specific involvement of GVHD. In summary, this article explores the potential of proteomics as a tool for timely and accurate intervention in the context of GVHD following allo-HSCT.
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Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Biomarcadores , Condicionamento Psicológico , ProteômicaRESUMO
The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.
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Criopreservação/métodos , Rafinose/farmacologia , Preservação do Sêmen/métodos , Ovinos , Espermatozoides , Taurina/análogos & derivados , Animais , Membrana Celular/efeitos dos fármacos , Criopreservação/veterinária , Dano ao DNA/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/efeitos dos fármacos , Taurina/farmacologiaRESUMO
Magnetic structures exhibiting large magnetic moments are sought after in theranostic approaches that combine magnetic hyperthermia treatment (MH) and diagnostic magnetic resonance imaging in oncology, since they offer an enhanced magnetic response to an external magnetic field. We report on the synthesized production of a core-shell magnetic structure using two types of magnetite nanoclusters (MNC) based on a magnetite core and polymer shell. This was achieved through an in situ solvothermal process, using, for the first time, 3,4-dihydroxybenzhydrazide (DHBH) and poly[3,4-dihydroxybenzhydrazide] (PDHBH) as stabilizers. Transmission electron microscopy (TEM) analysis showed the formation of spherical MNC, X-ray photoelectronic spectroscopy (XPS) and Fourier transformed infrared (FT-IR) analysis proved the existence of the polymer shell. Magnetization measurement showed saturation magnetization values of 50 emu/g for PDHBH@MNC and 60 emu/g for DHBH@MNC with very low coercive field and remanence, indicating that the MNC are in a superparamagnetic state at room temperature and are thus suitable for biomedical applications. MNCs were investigated in vitro, on human normal (dermal fibroblasts-BJ) and tumor (colon adenocarcinoma-CACO2, and melanoma-A375) cell lines, in view of toxicity, antitumor effectiveness and selectivity upon magnetic hyperthermia. MNCs exhibited good biocompatibility and were internalized by all cell lines (TEM), with minimal ultrastructural changes. By means of flowcytometry apoptosis detection, fluorimetry, spectrophotometry for mitochondrial membrane potential, oxidative stress, ELISA-caspases, and Western blot-p53 pathway, we show that MH efficiently induced apoptosis mostly via the membrane pathway and to a lower extent by the mitochondrial pathway, the latter mainly observed in melanoma. Contrarily, the apoptosis rate was above the toxicity limit in fibroblasts. Due to its coating, PDHBH@MNC showed selective antitumor efficacy and can be further used in theranostics since the PDHBH polymer provides multiple reaction sites for the attachment of therapeutic molecules.
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Cornus mas L. is characterized by an increased quantity of bioactive compounds, namely polyphenols, monoterpenes, organic acids, vitamin C and lipophilic compounds such as carotenoids, being anciently used in the treatment of various diseases. This paper's objectives were to characterize the phytochemical profile of Cornus mas L. fruits and to evaluate the in vitro antioxidant, antimicrobial and cytoprotective effects on renal cells exposed to gentamicin. As such, two ethanolic extracts were obtained. The resulting extracts were used to assess the total polyphenols, flavonoids and carotenoids through spectral and chromatographic methods. The antioxidant capacity was assessed using DPPH and FRAP assays. Due to the high content of phenolic compounds analyzed in fruits and the results obtained regarding antioxidant capacity, we decided to further use the ethanolic extract to investigate the in vitro antimicrobial and cytoprotective effects on renal cells stressed with gentamicin. The antimicrobial activity was assessed using agar well diffusion and broth microdilution methods, with great results regarding Pseudomonas aeruginosa. The cytotoxic activity was assessed using MTT and Annexin-V assays. According to the findings, extract-treated cells had a higher cell viability. However, at high concentrations, viability was shown to decline, most likely due to the extract and gentamicin's additive effects.
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Three individual hydroalcoholic extracts derived from Hamamelis virginiana leaves, Krameria lappacea root, Salix alba bark, and the resulting herbal mixture (HM) were assessed for the phytochemical profile as well as for antibacterial and cytotoxic potential. The chemical composition of the individual extracts and of their mixture was analyzed by chromatographical (LC-MS) and spectrophotometrical methods. The antimicrobial properties were evaluated by using the agar-well diffusion and the broth microdilution assays, whereas the potential cytotoxicity was investigated on human keratinocyte cell line by MTT method and apoptosis test. The HM composition revealed important amounts of valuable polyphenolic compounds provided from the individual extracts, having synergistic biological effects. All tested extracts displayed in vitro antimicrobial properties, with a significantly higher efficacy noticed for the HM when tested against Staphylococcus aureus. Moreover, none of the tested extracts was responsible for in vitro cytotoxicity against the human keratinocytes in the selected concentration range. Furthermore, the HM was included in an oil-in-water cream for the nonpharmacological treatment of seborrheic dermatitis, developed and optimized by using a QbD approach. A D-optimal experimental plan with four factors that varied on two levels was used to investigate the effect of the quantitative variation of the formulation factors (emulsifier, co-emulsifier, thickening agent, oily phase ratio) on the characteristics of the cream in terms of firmness, consistency, adhesiveness, stringiness, spreadability, and viscosity. Based on the experimental results, an optimal formulation containing 2.5% emulsifier and 20% oily phase was prepared and analyzed. The obtained results showed appropriate quality characteristics of this novel cream, which may be used in the future to manage the associated symptoms of seborrheic dermatitis.
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The most important concept behind using bone scaffolds is the biocompatibility of the material to avoid a local inflammatory response and must have the following properties: osteoinduction, osteoconductivity, angiogenesis, and mechanical support for cell growth. Gold nanoparticles/gold and silver nanoparticles -containing bioactive glasses in biopolymer composites have been used to enhance bone regeneration. These composites were testedin vitroon fibroblast and osteoblast cell lines using MTT tests, immunofluorescence, scanning electron microscopy analysis, andin vivoin an experimental bone defect in Sprague-Dawley rats. Both composites promoted adequate biological effects on human fibroblastic BJ (CRL 2522TM) cell lines and human osteoblastic cells isolated from the human patella in terms of cell proliferation, morphology, migration, and attachment. Most importantly, they did not cause cellular apoptosis and necrosis. According to the histological and immunohistochemical results, both composites were osteoinductive and promoted new bone formation at 60 d. Evidence from this study suggests that the small amount of silver content does not influence negatively thein vitroorin vivoresults. In addition, we obtained accurate results proving that the existence of apatite layer and proteins on the surface of the recovered composite, supports the validity ofin vitrobioactivity research.
Assuntos
Ouro , Nanopartículas Metálicas , Ratos , Animais , Humanos , Prata , Ratos Sprague-Dawley , Regeneração Óssea , Biopolímeros , Alicerces Teciduais/químicaRESUMO
BACKGROUND AND OBJECTIVE: Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder for which diagnosis is typically straightforward, based on bone marrow morphology and flow cytometry (FC) or immunohistochemistry. Nevertheless, variants present atypical expressions of cell surface markers, as is the case of CD5, for which the differential diagnosis can be more difficult. The aim of the current paper was to describe diagnosis of HCL with atypical CD5 expression, with an emphasis on FC. METHODS: The detailed diagnostic methodology for HCL with atypical CD5 expression is presented, including differential diagnosis from other lymphoproliferative diseases with similar pathologic features, by FC analysis of the bone marrow aspirate. RESULTS: Diagnosis of HCL by means of FC started by gating all events based on side scatter (SSC) versus CD45 and B lymphocytes were selected from the lymphocytes gate as CD45/CD19 positive. The gated cells were positive for CD25, CD11c, CD20, and CD103, while CD10 proved to be dim to negative. Moreover, cells positive for CD3, CD4, and CD8, the three pan-T markers, as well as CD19, showed a bright expression of CD5. The atypical CD5 expression is usually correlated with a negative prognosis and thus chemotherapy with cladribine should be initiated. CONCLUSION: HCL is an indolent chronic lymphoproliferative disorder and diagnosis is usually straightforward. However, atypical expression of CD5 renders its differential diagnosis more difficult, but FC is a useful tool that allows an optimal classification of the disease and allows initiation of timely satisfactory therapy.
Assuntos
Leucemia de Células Pilosas , Transtornos Linfoproliferativos , Humanos , Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/patologia , Citometria de Fluxo/métodos , Imunofenotipagem , Linfócitos B , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismoRESUMO
The research investigated the effect of gold (Au-CM) and silver nanoparticles (Ag-CM) phytoreduced with Cornus mas fruit extract (CM) on a human colorectal adenocarcinoma (DLD-1) cell line. The impact of nanoparticles on the viability of DLD-1 tumor cells and normal cells was evaluated. Oxidative stress and cell death mechanisms (annexin/propidium iodide analysis, caspase-3 and caspase-8 levels, p53, BCL-2, BAX, NFkB expressions) as well as proliferation markers (Ki-67, PCNA and MAPK) were evaluated in tumor cells. The nanoparticles were characterized using UV-Vis spectroscopy and transmission electron microscopy (TEM) and by measuring zeta potential, hydrodynamic diameter and polydispersity index (PDI). Energy dispersive X-ray (EDX) and X-ray powder diffraction (XRD) analyses were also performed. The nanoparticles induced apoptosis and necrosis of DLD-1 cells and reduced cell proliferation, especially Ag-CM, while on normal cells, both nanoparticles maintained their viability up to 80%. Ag-CM and Au-CM increased the expressions of p53 and NFkB in parallel with the downregulation of BCL-2 protein and induced the activation of caspase-8, suggesting the involvement of apoptosis in cell death. Lipid peroxidation triggered by Ag-CM was correlated with tumor cell necrosis rate. Both nanoparticles obtained with phytocompounds from the CM extract protected normal cells and induced the death of DLD-1 tumor cells, especially by apoptosis.