Carbenoxolone enhances inhibitory effect of RSL3 against cisplatin-resistant testicular cancer cells by promoting ferroptosis / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatinresistant testicular cancer cells (I-10/DDP) and the effect of carbenoxolone on the activity of RSL3 against testicular cancer.@*METHODS@#MTT assay was used to evaluate the survival rate of I-10/DDP cells following treatment with RSL3 (1, 2, 4, 8, 16 or 32 μmol/L) alone or in combination with carbenoxolone (100 μmol/L) or after treatment with Fer-1 (2 μmol/L), RSL3 (4 μmol/L), RSL3+Fer-1, RSL3+carbenoxolone (100 μmol/L), or RSL3+Fer-1+carbenoxolone. Colony formation assay was used to assess the proliferation ability of the treated cells; wounding-healing assay and Transwell assay were used to assess the invasion and migration ability of the cells. The expression of GPX4 was detected using Western blotting, the levels of lipid ROS were detected using C11 BODIPY 581/591 fluorescent probe, and the levels of Fe2+ were determined with FerroOrange fluorescent probe.@*RESULTS@#RSL3 dose-dependently decreased the survival rate of I-10/DDP cells, and the combined treatment with 2, 4, or 8 μmol/L RSL3 with carbenoxolone, as compared with RSL3 treatment alone, resulted in significant reduction of the cell survival rate. The combination with carbenoxolone significantly enhanced the inhibitory effect of RSL3 on colony formation, wound healing rate (P=0.005), invasion and migration of the cells (P < 0.001). Fer-1 obviously attenuated the inhibitory effects of RSL3 alone and its combination with carbenoxolone on I-10/DDP cells (P < 0.01). RSL3 treatment significantly decreased GPX4 expression (P=0.001) and increased lipid ROS level (P=0.001) and Fe2+ level in the cells, and these effects were further enhanced by the combined treatment with carbenoxolone (P < 0.01).@*CONCLUSION@#Carbenoxolone enhances the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatin-resistant testicular cancer cells by promoting RSL3-induced ferroptosis.

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Uso de marcadores fluorescentes na engenharia tecidual óssea: estudo piloto / Use of fluorescent markers in bone tissue engineering: pilot study

Introdução: a regeneração e o reparo de tecidos ósseos perdidos é objeto de estudo da Bioengenharia Tecidual. O uso de biomateriais substitutos ósseos biomiméticos visa estimular os sistemas celulares e bioquímicos para restabelecer de modo mais eficiente o tecido ósseo nos casos de sua reconstrução. Ao investigar o processo de remodelação, é vital identificar áreas de novo crescimento para avaliar a eficácia dos biomateriais implantados e respectivos regimes de tratamento. A avaliação qualitativa e quantitativa da regeneração óssea pode ser realizada através da aplicação de marcadores como o Xilenol, a Tetraciclina, a Calceína e a Alizarina. A administração desses marcadores de forma associada possibilita ainda marcar sequencialmente camadas de nova deposição e remodelação durante o reparo. Objetivo: estabelecer um protocolo para utilização dos marcadores fluorescentes de reparo ósseo xilenol, tetraciclina, calceína e alizarina, em ratos. Metodologia: foram utilizados 35 ratos da linhagem Wistar, machos adultos, com massa corpórea entre 350 e 400g, e idade aproximada de 4 a 5 meses, distribuídos randomicamente em 5 grupos experimentais, submetidos à confecção de defeito ósseo circular de 8 mm em região de calvária, e administração dos diferentes marcadores segundo os grupos; XO ­ Xilenol; Ca ­ Calceína; Al ­ Alizarina; Te ­ Tetraciclina; C ­ Controle. Após 15 dias de experimento, os animais foram eutanasiados e as calvárias processadas e analisadas por histomorfometria, microscopia de epifluorescência e microscopia de fluorescência. Resultados: todos protocolos empregados para utilização dos marcadores fluorescentes xilenol, calceína, alizarina e tetracicilina foram úteis para identificar área de deposição mineral durante o período analisado de regeneração óssea em ratos. As imagens obtidas pela microscopia de fluorescência revela a presença dos marcadores incorporados à matriz óssea neoformada, no entanto a utilização da Alizarina e Calceína dentro dos protocolos testados mostraram-se mais eficientes. Conclusão: os protocolos testados nesse estudo apresentaram-se viáveis para utilização em pesquisas envolvendo marcadores de regeneração óssea, com resultados superiores para Alizarina e Calceína

Introduction: The regeneration and repair of lost bone tissues is the subject of a study of Tissue Bioengineering. The use of biomimetic biomaterial bone substitutes aims to stimulate the cellular and biochemical systems to restore more efficiently the bone tissue in the cases of its reconstruction. When investigating the remodeling process, it is vital to identify areas of new growth to evaluate the efficacy of implanted biomaterials and their treatment regimens. The qualitative and quantitative evaluation of bone regeneration can be performed through the use of markers such as Xylenol, Tetracycline, Calcein and Alizarin. The administration of such markers in an associated manner also makes it possible to sequentially mark layers of new deposition and remodeling during the repair. Objective: to establish a protocol for the use of fluorescent xylenol, tetracycline, calcein and alizarin bone repair markers in rats. Metodology: thirtyfive male adult Wistar rats with a body mass ranging from 350 to 400 g and approximately 4 to 5 months old were randomly assigned to 5 experimental groups submitted to a circular bone defect of 8 mm in the region of calvaria, and administration of the different markers according to the groups; XO ­ Xylenol; Ca ­ Calcein; Al-Alizarin; Te ­ Tetracycline; C ­ Control. After 15 days of experiment, the animals were euthanized and the calvaria processed and analyzed by histomorphometry, epifluorescence microscopy and fluorescence microscopy. Results: all protocols used for fluorescence markers xylenol, calcein, alizarin and tetracycline were useful to identify area of mineral deposition during the analyzed period of bone regeneration in rats. The images obtained by fluorescence microscopy revealed the presence of the markers incorporated into the neoformed bone matrix, however the use of Alizarin and Calcein within the protocols tested were more efficient. Conclusion: the protocols tested in this study were feasible for use in research involving markers of bone regeneration, with superior results for Alizarin and Calcein.

Distribution and accumulation of Cy5.5-labeled thermally cross-linked superparamagnetic iron oxide nanoparticles in the tissues of ICR mice

Free Cy5.5 dye and Cy5.5-labeled thermally cross-linked superparamagnetic iron oxide nanoparticles (TCL-SPION) have been routinely used for in vivo optical imaging. However, there is little information about the distribution and accumulation of free Cy5.5 dye and Cy5.5-labeled TCL-SPION in the tissues of mice. Free Cy5.5 dye (0.1 mg/kg body weight) and Cy5.5-labeled TCL-SPION (15 mg/kg body weight) were intravenously injected into the tail vein of ICR mice. The biodistribution and accumulation of the TCL-SPION and Cy5.5 were observed by ex vivo optical imaging and fluorescence signal generation at various time points over 28 days. Cy5.5 dye fluorescence in various organs was rapidly eliminated from 0.5 to 24 h post-injection. Fluorescence intensity of Cy5.5 dye in the liver, lung, kidney, and stomach was fairly strong at the early time points within 1 day post-injection. Cy5.5-labeled TCL-SPION had the highest fluorescence density in the lung at 0.5 h post-injection and decreased rapidly over time. Fluorescence density in liver and spleen was maintained over 28 days. These results suggest that TCL-SPION can be useful as a carrier of therapeutic reagents to treat diseases by persisting for long periods of time in the body.

Knockdown of apoptosis-inducing factor disrupts function of respiratory complex I

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.

Preliminary Effects of Oral Uridine on the Ocular Surface in Dry Eye Patients

We designed a randomized, double blinded, 3-months controlled prospective clinical study to investigate effects of oral uridine on the ocular surface in dry eye patients. Twenty-seven patients who diagnosed as dry eye with lower than 5 mm of wetting in the Schirmer strip, with corneal epithelial erosion and who completely followed-up till 3 months were enrolled. Corneal-conjunctival fluorescein staining, non-anesthetic Schirmer test, impression cytology, and Ocular Surface Disease Index (OSDI) were evaluated in the experimental and placebo groups at the baseline, 1 and 3 months after start of medication in a double blinded manner. Fluorescein stain score of the cornea was markedly decreased in oral uridine group compared to the placebo group at 3 months after medication (P=0.032, Mann-Whitney U test). The Schirmer wetting score for the oral uridine group was significantly increased (P=0.001, Wilcoxon signed rank test) at 3 months and its difference between two groups was statistically significant (P=0.030, Mann-Whitney U test). OSDI scores were significantly decreased at 1 and 3 months in treatment group. Oral uridine is effective in treatment of dry eyes.

Imaging of biophoton emission from electrostimulated skin acupuncture point jg4: effect of light enhancers.

Using an ultrasensitive CCD camera, an extremely low light intensity from the acupuncture-sensitive point JG4 at the left hand was recorded. As the intensity of the light was very weak and the time of electrostimulation exceeded the recommended period, the quality of biophoton images was poor. Chemiluminescent and fluorescent hydrophilic, hydrophobic and amphyphilic molecular probes were used to: (i) ensure penetration of probes into skin, (ii) enhance the intensity of BP emission, (iii) shorten time and (iv) obtain information about mechanisms of biophotons generation in EAP-sensitive points and channels. The results obtained partially fulfilled expectations and indicate on the necessity to elaborate special techniques of probes deposition on the skin.

A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation

Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.

GC-rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces).

In a bid to ascertain the molecular architecture of the silver positive regions (NORs) in chromosomes of three species of fish, namely, Hemibagrus menoda (Hamilton), Sperata seenghala (Sykes) (Fam: Bagridae) and Mastacembelus armatus (Lacep6de) (Fam: Mastacembelidae), an additional staining methodology using a fluorochrome dye (Chromomycin A3) was deployed along with the AgNO3 technique. The nucleolar organizing regions (NORs) were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes (No.3) in H. menoda (2n=58), at the longer arms (Tq) of one pair of submetacentric chromosomes (No.5) in S. seenghala (2n=50) and at the shorter arm (Tp) of one pair of homologous submetacentric chromosomes (No.6) in M. armatus (2n=48). Staining with Chromomycin A3 produced bright fluorescing zones in GC-rich heterochromatin of Ag-positive NORs. The results indicate a more general trend of existence of an overlapping region between NOR and GC-rich fluorescing zones, the active sites of rRNA genes (rDNA) in this primitive group of vertebrates although exceptions to this situation has been reported in a couple of extant fish species earlier. More data utilizing such combined methodologies are warranted to understand the structural organization of fish chromosomes more precisely.

Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide

To ascertain whether used and re-refined lubricant oil absorbed through the skin can produce a genotoxic effect or cytotoxicity in mouse bone marrow cells, we examined the induction of micronucleated erythrocytes of peripheral blood after cutaneous application. Both re-refined and used lubricant oils showed a weak but significant induction of micronucleated polychromatic erythrocytes compared with control, while virgin oil did not show micronucleus induction. Cyclophosphamide (CP) was used not only as positive control but also to compare the sensitivity between intraperitoneal and dermal routes of administration of the test compounds in mice. The efficacy of intraperitoneal injection of CP is well known. On the other hand, dermal exposure is not so common and when CP was diluted in glycerin statistically significant values (P = 0.0036) of micronuclei were also found. Topically applied lubricant oils (virgin, re-refined and used) have the capacity to interfere with mouse bone marrow hematopoiesis evidenced by a statistically significant decrease in the proportion of polychromatic erythrocytes in the peripheral blood. Physical and chemical analysis revealed that used oil is more viscous than other lubricants, suggesting the presence of insoluble compounds, oxidized products and water as well as aromatic hydrocarbons. Used oil differs from other lubricant oils in metal and polyaromatic hydrocarbon content. Re-refined oil revealed a neutral value typical of pure mineral oil. This assay is an important tool to evaluate environmental pollutants that cause genotoxicity and/or cytotoxicity through skin exposure.

Rapid Detection of Duplication/Deletion of the PMP22 Gene in Patients with Charcot-Marie-Tooth Disease Type 1A and Hereditary Neuropathy with Liability to Pressure Palsy by Real-time Quantitative PCR using SYBR Green I Dye

Mutations and altered gene dosage of the peripheral myelin protein (PMP22) gene in chromosome 17p11.2-12 are the main causes for hereditary neuropathies, accounting for approximately 70% of all cases. Patients with duplication of the PMP22 develop Charcot-Marie-Tooth disease type 1A (CMT1A) and deletion of one PMP22 allele leads to hereditary neuropathy with liability to pressure palsy (HNPP). Twenty patients with CMT1A, 17 patients with HNPP, and 18 normal family members and 28 normal controls were studied by real-time quantitative PCR using SYBR Green I on the ABI 7700 Sequence Detection System. The copy number of the PMP22 gene was determined by the comparative threshold cycle method and the albumin was used as a reference gene. The PMP22 duplication ratio ranged from 1.45 to 2.06 and the PMP22 deletion ratio ranged from 0.42 to 0.64. The PMP22 ratio in normal controls, including normal family members, ranged from 0.85 to 1.26. No overlap was found between patients with CMT1A or patients with HNPP and normal controls. This method is fast, highly sensitive, specific, and reproducible in detecting PMP22 duplication and deletion in CMT1A and HNPP patients, respectively.

Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 æm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 æg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion

The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.

Tomometria de aplanaçäo com e sem fluoresceína / Applanation tonometry with and without fluorescein

A técnica original da tonometria de aplanaçäo, descrita por Goldmann e Schmidt, recomenda o uso da fluoresceína no saco conjuntival. A maioria dos oftamologistas faz uso rotineiro da fluoresceína, embora alguns a considere dispensável. Para esclarer essa controvérsia, foi realizado este trabalho, submetendo-se 194 pacientes á tonometria de aplanaçäo com ou sem fluoresceína, para se verificar a diferença entre dois procedimentos.