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This study reviews recent progress in the surgical treatment of Rathke's cleft cysts (RCCs) and Sellar region meningiomas, based on findings from three key studies. RCCs are benign, fluid-filled remnants from pituitary gland development that are usually asymptomatic and found by chance. However, surgical intervention is needed when they become symptomatic or increase in size. Research by Stefan Linsler et al. and others examines various surgical methods, including transcranial keyhole and transsphenoidal techniques for RCCs, and endoscopic endonasal and supraorbital keyhole approaches for Sellar meningiomas. The results show that both transcranial keyhole and transsphenoidal surgeries for RCCs have high success rates with no recurrences over 5.7 years, although the keyhole approach has fewer complications. For Sellar meningiomas, the choice between endoscopic endonasal and supraorbital keyhole techniques should be based on tumor characteristics, highlighting the importance of surgeon proficiency in both methods. These studies emphasize the need for personalized treatment strategies tailored to patient and tumor characteristics and highlight the importance of ongoing surgical skill development and further research to refine minimally invasive techniques. This study highlights the crucial role of personalized surgical approaches in improving outcomes for patients with RCCs and Sellar region meningiomas.
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Cistos do Sistema Nervoso Central , Neoplasias Meníngeas , Meningioma , Procedimentos Neurocirúrgicos , Sela Túrcica , Humanos , Meningioma/cirurgia , Cistos do Sistema Nervoso Central/cirurgia , Procedimentos Neurocirúrgicos/métodos , Neoplasias Meníngeas/cirurgia , Sela Túrcica/cirurgia , Neoplasias Hipofisárias/cirurgia , Resultado do Tratamento , Neuroendoscopia/métodosRESUMO
Exposure to microgravity can adversely affect the fitness of astronauts. The integrity of the skin plays a crucial role in protecting against mechanical forces and infections, fluid imbalance, and thermal dysregulation. In brief, the skin wound may cause unknown challenges to the implementation of space missions. Wound healing is a physiological process that relies on the synergistic action of inflammatory cells, extracellular matrix (ECM), and various growth factors to maintain the integrity of skin after trauma. Fibroblasts are present almost throughout the entire process of wound repair, especially in the scar formation at the endpoint of wound healing. However, there is limited knowledge about the extent to which fibroblasts are affected by the lack of gravity during wound healing. In this study, we utilized the rotary cell culture system, a ground-based facility that mimics the weightless condition, to study the alterations of L929 fibroblast cells under simulated microgravity (SMG). Our results demonstrated that the SM condition exerted negative influences on the proliferation and ECM formation of the L929 fibroblast. Whereas, the apoptosis of fibroblast was significantly upregulated upon exposure to SMG conditions. Moreover, the transforming growth factor-ß1/Smad3 (TGF-ß1/smad3) signaling pathway of L929 fibroblast related to wound repair was also altered significantly under a weightless environment. Overall, our study provided evidence that fibroblasts are strongly sensitive to SMG and elucidated the potential value of the TGF-ß1/Smad3 signaling pathway modulating wound healing in the future practice of space medicine.
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Fator de Crescimento Transformador beta1 , Ausência de Peso , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Transdução de Sinais , Matriz Extracelular , Apoptose , Proliferação de Células , Fibroblastos/metabolismo , Proteína Smad3/metabolismoRESUMO
BACKGROUND: Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice. MATERIALS AND METHODS: To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production. RESULTS: Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts. CONCLUSIONS: Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.
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Carcinoma de Células Renais , Neoplasias Renais , Preservação de Sangue , Criança , Eritrócitos/efeitos da radiação , Raios gama , Hemólise , Humanos , Recém-Nascido , Potássio , Fatores de TempoRESUMO
Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants' response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.
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Fabaceae , Rhizobium , Fabaceae/metabolismo , Gliceraldeído , Fixação de Nitrogênio , Pressão Osmótica , Pisum sativum/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Rhizobium/metabolismo , Nódulos Radiculares de Plantas/metabolismo , SimbioseRESUMO
BACKGROUND: Indian hedgehog (IHH) and Sonic hedgehog (SHH) are important regulators of chondrogenesis. However, activation of IHH and SHH also promotes chondrocyte hypertrophy and ossification during chondrogenesis. The aims of this study were to investigate the effect of microgravity on IHH- and SHH-induced chondrogenic differentiation and to elucidate the role of microgravity in this process. METHODS: Adenovirus plasmids encoding the rabbit IHH gene and SHH genes were constructed in vitro and transfected into rabbit bone marrow-derived mesenchymal stem cells (BMSCs). A rotary cell culture system (RCCS), in which a dynamic three-dimensional culture system combines the mechanical environment with a three-dimensional culture surface, was used for cell culture and differentiation. During the induction of differentiation, expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Toluidine blue and collagen II immunohistochemical staining and annexin V-Cy3 staining were used to indicate investigate cartilage matrix synthesis and hypertrophic hypertrophy, respectively, on day 21 after induction of differentiation. RESULTS: In this study, IHH and SHH were shown to be equipotent inducers of chondrogenesis in rabbit BMSCs, as evidenced by strong staining for proteoglycans and collagen II, and increased expression of mRNAs and proteins associated with chondrogenesis in an RCCS environment. More importantly, chondrogenic hypertrophy and aging were effectively inhibited in the RCCS environment. In addition, levels of cartilage-related markers in the IHH and SHH transfection groups were initially increased and later decreased in the traditional two-dimensional environment, while cartilage hypertrophy-related factors revealed higher mRNA expression levels during induction. CONCLUSIONS: In summary, microgravity significantly promoted chondrogenic differentiation of BMSCs induced by IHH and SHH and attenuated chondrogenic hypertrophy and aging during chondrogenesis. Furthermore, exogenous IHH and SHH had the same effect on chondrogenic differentiation of BMSCs in the RCCS environment. This study provides further evidence of chondrogenic induction of BMSCs in vitro via IHH and SHH gene delivery.
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Medula Óssea/metabolismo , Condrogênese , Proteínas Hedgehog/fisiologia , Células-Tronco Mesenquimais/metabolismo , Animais , Medula Óssea/fisiologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Mesenquimais/fisiologia , Coelhos , Transdução de SinaisRESUMO
Microgravity (µXg) experienced by astronauts during space flights causes accelerated bone loss. However, the molecular basis of µXg induced bone loss in space is unclear. Osteoclast (OCL) is the primary bone-resorbing cell. We previously demonstrated that simulated µXg promotes OCL formation. In this study, we identified that µXg induces syncytin-A expression in RAW264.7 preosteoclast cells without RANKL stimulation. We further tested the effect of osteotropic factors such as CXCL5 and 1,25(OH)2 D3 to regulate the syncytin-A expression in preosteoclast cells subjected to µXg compared to ground based (Xg) cultures. CXCL5 (25 ng/mL) and 1,25(OH)2 D3 (10 ng/mL) increased syncytin-A expression under Xg conditions. However, µXg alone upregulates syncytin-A expression compared to Xg control preosteoclast cells. Confocal microscopy using Lyso-Tracker identified syncytin-A expression co-localized with lysosomes in preosteoclast cells. Acridine orange staining showed RANKL elevated autophagy activity in these cells. Further, siRNA suppression of syncytin-A significantly inhibits autophagy activity in RAW264.7 cells. In addition, knockdown of syncytin-A expression inhibits µXg increased OCL formation in mouse bone marrow cultures. Thus, our findings suggest that targeting syncytin-A expression may be an effective countermeasure to control bone loss under microgravity conditions.
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Diferenciação Celular , Osteoclastos/citologia , Osteogênese , Proteínas da Gravidez/metabolismo , Ausência de Peso , Animais , Autofagia , Camundongos , Osteoclastos/metabolismo , Proteínas da Gravidez/genética , Células RAW 264.7 , Voo EspacialRESUMO
Multiple myeloma is an aggressive tumour able to suppress osteoblastogenesis probably mediated by bone marrow mesenchymal stromal cells (BM-MSCs) that can also support plasma cell growth/survival. The use of MSCs for multiple myeloma therapy is a controversial topic because of the contradictory results on the capacity of MSCs to inhibit or to promote cancer growth. Our previous studies demonstrated that MSCs could be loaded with Paclitaxel (PTX) and used to deliver the drug in situ in amount affecting tumour growth (in vitro and in vivo). Therefore, independently on the discussed action of MSCs in myeloma, MSCs could represent a 'trojan horse' to vehicle and deliver anti-tumour agents into bone marrow. This study confirms, by an in vitro 3D dynamic culture system, that PTX loaded BM-MSCs (PTXr-MSCs) are active on the proliferation of RPMI 8226, a human myeloma cell line. Our results demonstrated a dramatic suppression of myeloma cell growth by PTXr-MSCs, suggesting that drug loaded MSCs could be a tool to deliver drug into the bone marrow. Drug releasing MSCs provide a therapeutic approach to potentiate the existing treatments against a very aggressive malignancy as multiple myeloma. Copyright © 2016 John Wiley & Sons, Ltd.
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Antineoplásicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/metabolismo , Paclitaxel/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultivo Condicionados , Resistencia a Medicamentos Antineoplásicos , Tolerância a Medicamentos , Humanos , Mieloma Múltiplo/patologia , Esferoides Celulares , Células Tumorais CultivadasRESUMO
BACKGROUND AND OBJECTIVE: Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. MATERIAL AND METHODS: After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. RESULTS: Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. CONCLUSION: For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications.
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Alginatos , Materiais Biocompatíveis , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Alicerces Teciduais , Adipogenia/fisiologia , Alginatos/química , Apatitas/análise , Materiais Biocompatíveis/química , Reatores Biológicos , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Condrogênese/fisiologia , Colágeno Tipo I/análise , Cadeia alfa 1 do Colágeno Tipo I , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Matriz Extracelular/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Hidrodinâmica , Microesferas , Propriedades de Superfície , Alicerces Teciduais/química , Geleia de Wharton/citologiaRESUMO
Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5-100µM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca(2+) overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca(2+) overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.
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Catequina/análogos & derivados , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Chá/química , Animais , Western Blotting , Catequina/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Masculino , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Fosforilação Oxidativa/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Coloração pela PrataRESUMO
Long-term spaceflights affect the structural changes in brain, alter motor or cognitive function and associated development of neuro-optic syndrome in astronauts. Studies addressing the impact of microgravity on brain cells are very limited. Herein, we employed microglial (CHME3) and glioblastoma (U87MG and A172) cells to study their molecular and functional adaptations under simulated microgravity (SMG) exposure. A reduction in cell viability and proliferation with decreased levels of PCNA were observed in these cells. SMG caused extensive DNA damage with an increase in γH2A.X (ser139) phosphorylation and differential activation/expression of DNA damage response (DDR) proteins including ATM, ATR, Chk1, Chk2 and p53 in all the three cell lines. Unlike CHME3, the ATM/Chk2-dependent DDR pathway was activated in glioblastoma cells suggesting a marked difference in the adaptation between normal and cancer cells to SMG. Five different classes of DNA repair pathways including BER, NER, MMR, NHEJ and HR were suppressed in both cell lines with the notable exception of NHEJ (Ku70/80 and DNA-PK) activation in U87MG cells. SMG induced mitochondrial apoptosis with increased expression of Bax, cleaved caspase-3 and cleaved poly-(ADP-ribose) polymerase, and reduced Bcl-2 level. SMG triggered apoptosis simultaneously via ERK1/2 and AKT activation, and inhibition of GSK3ß activity which was reversed by MEK1 and PI3K inhibitors. Taken together, our study revealed that microgravity is a strong stressor to trigger DNA damage and apoptosis through activation of ERK1/2 and AKT, and impairment of DNA repair capacity, albeit with a cell-type difference in DDR and NHEJ regulation, in microglial and glioblastoma cells.
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Glioblastoma , Ausência de Peso , Humanos , Glioblastoma/genética , Microglia , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Reparo do DNA , Dano ao DNARESUMO
In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.
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Biofilmes , Mycobacterium bovis , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/fisiologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Objectives: Public sharing of de-identified biomedical data promotes collaboration between researchers and accelerates the development of disease prevention and treatment strategies. However, open-access data sharing presents challenges to researchers who need to protect the privacy of study participants, ensure that data are used appropriately, and acknowledge the inputs of all involved researchers. This article presents an approach to data sharing which addresses the above challenges by using a publicly available dashboard with de-identified, aggregated participant data from a large HIV surveillance cohort. Materials and Methods: Data in this study originated from the Rakai Community Cohort Study (RCCS), which was integrated into a centralized data mart as part of a larger data management strategy for the Rakai Health Sciences Program in Uganda. These data were used to build a publicly available, protected health information (PHI)-secured visualization dashboard for general research use. Results: Using two unique case studies, we demonstrate the capability of the dashboard to generate the following hypotheses: firstly, that HIV prevention strategies ART and circumcision have differing levels of impact depending on the marital status of investigated communities; secondly, that ART is very successful in comparison to circumcision as an interventional strategy in certain communities. Discussion: The democratization of large-scale anonymized epidemiological data using public-facing dashboards has multiple benefits, including facilitated exploration of research data and increased reproducibility of research findings. Conclusion: By allowing the public to explore data in depth and form new hypotheses, public-facing dashboard platforms have significant potential to generate new relationships and collaborations and further scientific discovery and reproducibility.
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BACKGROUND AND AIMS: Subpopulations of immature red blood cells (RBCs) named CD71+ erythroid cells (CECs) with different properties may contribute to RBC transfusion outcomes. However, it is challenging to quantify CECs in leukoreduced RCCs and whole blood due to their rarity and fragility. Current flow cytometry methods are not applicable to leukoreduced RCCs as there is limited peripheral blood mononuclear cells (PBMCs) to use for determination of CECs. We have developed and validated a flow cytometry method for quantifying CECs in WB. METHODS: We determined optimal PE-Cy7-CD235a, BV711-CD71, APC-CD45 concentrations and instrument setting by titration. Linearity and level of detection was determined by spiking labelled PBMCs from cord blood units into unlabeled WB. Low, medium, high levels of CECs were used to determine intra- and inter -run precision. RESULTS: Detection of CECs was linear (R2 = 0.98) over the range of concentrations assessed with a limit of detection of 0.005%. The overall CVs for the intra- and inter-run precision were 6.9% and 9.7%. CONCLUSION: We developed a simple, sensitive, and cost-effective flow cytometric method for quantifying the proportion of CECs in non-manipulated WB, which could help understand the impact of RBC products on recipient transfusion outcomes.
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Eritrócitos , Leucócitos Mononucleares , Contagem de Células , Contagem de Eritrócitos , Citometria de Fluxo , HumanosRESUMO
Since humans started to explore the possibilities of life beyond earth, space missions began unmanned in the beginning and late with astronauts to explore the curiosity. There are several factors in space which are different from that of the earth's atmosphere, including gravity, cosmic radiation, extreme temperatures, and heavy ions. The space environment consists of very less gravitational force, which is often termed as microgravity. Microgravity, along with radiation in space, is known to suppress the immune system of astronauts, and hence, the chances of getting diagnosed with cancer could not be overlooked. Microgravity simulators are used to generate microgravity in laboratory conditions, and it could be a very beneficial tool to study the effect of altered gravity on various types of cells, including cancer cells. Cancer cells produce several biomarkers which are used for cancer diagnosis and prognosis. Protein biomarkers are indispensable for the ongoing fight against cancer. In this chapter, we describe the culture of both anchorage-dependent and suspension cell lines using rotary cell culture system (RCCS) to induce microgravity condition and its effect on protein biomarkers.
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Neoplasias , Ausência de Peso , Biomarcadores Tumorais , Técnicas de Cultura de Células , Humanos , Sistema Imunitário , Neoplasias/diagnóstico , Ausência de Peso/efeitos adversos , Simulação de Ausência de PesoRESUMO
Of all primary renal neoplasms, 80-85% are renal cell carcinomas (RCCs), which develop in the renal cortex. There are more than 10 histological and molecular subtypes of the disease, the most frequent of which is clear cell RCC, which also causes most cancer-related deaths. Other renal neoplasms, including urothelial carcinoma, Wilms' tumor, and renal sarcoma, each affect a particular age group and have specific gross and histological features. Due to the genetic susceptibility of each of these malignancies, early mutation discovery is necessary for the early detection of a tumor. Furthermore, it is crucial to avoid environmental factors leading to each type. This study provides relatively detailed and essential information regarding each subtype of renal carcinoma.
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The Rotary Cell Culture System (RCCS) is an apparatus that was originally designed by NASA engineers to simulate microgravity conditions for growth of both eukaryotic and bacterial cell cultures. The RCCS growth environment is also characterized by low fluid shear stress, thereby also providing an in vitro growth condition relevant to certain in vivo environments encountered during bacterial infection. This chapter describes a method for growing Staphylococcus aureus under simulated microgravity conditions using the RCCS and disposable High Aspect Ratio Vessels (HARVs). Small samples can be removed and replaced with fresh media during the experiment (continuous sampling method) or the whole culture can be removed at the end of the experiment (end-point sampling method) for larger sample volumes required for follow-up studies such as RNAseq or proteomics.
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Técnicas Bacteriológicas/métodos , Staphylococcus aureus/crescimento & desenvolvimento , Simulação de Ausência de Peso/instrumentação , Técnicas Bacteriológicas/instrumentação , Perfilação da Expressão Gênica , Proteômica , Análise de Sequência de RNA , Resistência ao CisalhamentoRESUMO
BACKGROUND: Recently, neural stem cell (NSC) therapy has shown promise for the treatment of many neurological diseases. Enhancing the quality of implanted cells and improving therapeutic efficacy are currently research hotspots. It has been reported that collagen sponge material provided sufficient room for cell growth in all directions and promoted the absorption of nutrients and removal of wastes. And also, the Rotary Cell Culture System (RCCS), which mimics the microgravity environment, can be used to culture cells for tissue engineering. MATERIALS AND METHODS: We performed the mRNA and miRNA sequencing to elucidate the regulatory mechanism of NSCs cultured on the collagen sponge in the RCCS system. The luciferase assay and Western blot revealed a direct regulatory role between let-7i-5p and neurotrophic receptor tyrosine kinase 3 (NTRK3; also called TrkC). And then, the neural differentiation markers Tuj1 and Map2 were detected by immunofluorescence staining. In the meantime, the migratory ability of NSCs was detected both in vitro and in spinal cord injury animals. RESULTS: In this study, we demonstrated that the expression of NTRK3 was elevated in NSCs cultured on collagen sponge in the RCCS system. Furthermore, increased NTRK3 expression was regulated by the downregulation of let-7i-5p. Compared to traditionally cultured NSCs, the NSCs cultured on collagen sponge in the RCCS system exhibited better neuronal differentiation and migratory ability, especially in the presence of NT-3. CONCLUSIONS: As the biological properties and quality of transplanted cells are critical for therapeutic success, the RCCS system combined with the collagen sponge culture system shows promise for applications in clinical practice in the future.
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Células-Tronco Neurais , Alicerces Teciduais , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Colágeno , Receptor trkCRESUMO
BACKGROUND: It was confirmed that simulated microgravity (SMG) led to ultrastructural alterations and apoptosis in many types of microvascular endothelial cells. However, whether SMG would also affect choroidal vascular endothelial cells (CVECs) remains unknown. This study was designed to investigate the effects of SMG on ultrastructure and apoptosis of CVECs. METHODS: The rotary cell culture system (RCCS) was utilized to simulate microgravity condition. Human CVECs were cultured under normal gravity (NG) or SMG condition for 3 days. The ultrastructure was viewed under transmission electron microscopy, and the organization of F-actin was observed by immunofluorescence staining. Additionally, the apoptosis percentage was calculated using flow cytometry. Moreover, the mRNA and protein expression of BAX, Bcl-2, Caspase3, Cytochrome C, p-AKT, and p-PI3K were detected with quantitative PCR and Western blot at different exposure time. RESULTS: In the SMG group, CVECs presented with a shrunk cell body, chromatin condensation and margination, mitochondria vacuolization, and apoptotic bodies. The amount of F-actin decreased, and the filaments of F-actin were sparse or even partly discontinuous after cultivation under SMG for 72 h. The proportions of apoptotic CVECs in SMG groups at 24 and 72 h were significantly higher than those in the NG group (P < 0.001). The mRNA and protein expression of Bax, Caspase3, and Cytochrome C of CVECs in SMG groups at 24 and 72 h significantly increased than those of the NG group, respectively (P < 0.001). The alterations of p-AKT and p-PI3K protein expression possessed similar trends. On the contrary, the mRNA and protein expression of Bcl-2 in CVECs under SMG at 24 and 72 h were significantly less than that of the NG group, respectively (P < 0.001). CONCLUSION: Simulated microgravity conditions can lead the alterations of the F-actin structure and apoptosis of CVECs. The Bcl-2 apoptosis pathway and PI3K/AKT pathway may participate in the damage of CVECs caused by SMG.
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Simulated microgravity can significantly affect various cell types and multiple systems of the human body, such as cardiovascular system, skeletal muscle system, and immune system, and is known to cause anemia and loss of electrolyte and fluids. Epidermal stem cells (EpSCs) were cultured in a rotary cell culture system (RCCS) bioreactor to simulate microgravity. The metabolites of EpSCs were identified by liquid chromatography-mass spectrometry (LC-MS). Compared with normal gravity (NG) group, a total of 57 different metabolites of EpSCs were identified (P < 0.05, VIP > 1), including lipids and lipid-like molecules (51 molecules), amino acids (5 molecules), nucleosides, nucleotides, and analogues (1 molecule). According to the partial least squares discriminant analysis (PLS-DA) score plot, a VIP > 1 and P < 0.05 were obtained for the 57 different metabolites, of which 23 molecules were significantly downregulated and 34 were significantly upregulated in simulated microgravity (SMG) group. These results showed that SMG has a significant impact on different pathways, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that multiple pathways were involved, mainly the amino acid metabolism pathway, lipid metabolism pathway, membrane transport pathway, and cell growth and death pathways. Thus, the metabolic profile of EpSCs was changed under SMG. Exploring the metabolic profile of EpSCs would be helpful to further understand the growth characteristics of EpSCs under SMG, which will provide a new approach to explore the metabolomics mechanism of stress injury and repair trauma under SMG.
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Células Epidérmicas/metabolismo , Células-Tronco/metabolismo , Simulação de Ausência de Peso , Células Cultivadas , Gravitação , Humanos , Metabolismo dos Lipídeos , Metaboloma , MetabolômicaRESUMO
Oxidative stress is ubiquitous in environmental stresses and prevails over the cellular metabolic and phenotypic responses in plants. Reactive oxygen species (ROS) generated under stress affect macromolecules to form another group of toxic compounds called reactive carbonyl compounds (RCCs). These molecules have a longer half-life than ROS and cause carbonyl stress that affects cellular metabolism, cellular homeostasis, and crop productivity. The later effect of oxidative stress in terms of the generation of RCCs and glycation products and their effects on plant processes have not been explored well in plant biology. Therefore, how these molecules are produced and a few important effects of RCCs on plants have been discussed in this review article. Further, the plant adaptive detoxification mechanisms of RCCs have been discussed. The enzymes that were identified in plants to detoxify these cytotoxic compounds have broad substrate specificity and the potential for use in breeding programs. The review should provide a comprehensive understanding of the cytotoxic compounds beyond ROS and subsequently their mitigation strategies for crop improvement programs.