Synergistic effect of human uterine cervical mesenchymal stem cell secretome and paclitaxel on triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer and, despite its adverse effects, chemotherapy is the standard systemic treatment option for TNBC. Since, it is of utmost importance to consider the combination of different agents to achieve greater efficacy and curability potential, MSC secretome is a possible innovative alternative. METHODS: In the present study, we proposed to investigate the anti-tumor effect of the combination of a chemical agent (paclitaxel) with a complex biological product, secretome derived from human Uterine Cervical Stem cells (CM-hUCESC) in TNBC. RESULTS: The combination of paclitaxel and CM-hUCESC decreased cell proliferation and invasiveness of tumor cells and induced apoptosis in vitro (MDA-MB-231 and/or primary tumor cells). The anti-tumor effect was confirmed in a mouse tumor xenograft model showing that the combination of both products has a significant effect in reducing tumor growth. Also, pre-conditioning hUCESC with a sub-lethal dose of paclitaxel enhances the effect of its secretome and in combination with paclitaxel reduced significantly tumor growth and even allows to diminish the dose of paclitaxel in vivo. This effect is in part due to the action of extracellular vesicles (EVs) derived from CM-hUCESC and soluble factors, such as TIMP-1 and - 2. CONCLUSIONS: In conclusion, our data demonstrate the synergistic effect of the combination of CM-hUCESC with paclitaxel on TNBC and opens an opportunity to reduce the dose of the chemotherapeutic agents, which may decrease chemotherapy-related toxicity.

Novel bioanalytical strategy using isotope pattern deconvolution and ICP-QMS for the study of iron incorporation in erythrocytes: An insight to better assessment.

Iron is an essential element for human life and its nutritional status in the human body is directly linked to human health. More than 1015 atoms of iron per second are necessary for the maintenance of haemoglobin formation. To predict iron bioavailability three approaches are normally employed: (a) faecal recovery; (b) plasma appearance; and (c) erythrocyte incorporation (the most used). Isotope Pattern Deconvolution (IPD) is a mathematical tool that allows the isolation of distinct isotope signatures from mixtures of natural abundance and enriched tracers. In this work we propose a novel strategy to assess erythrocyte iron incorporation, based on the use of an iron stable isotope (57Fe) and the IPD concept. This strategy allows direct calculation of the exogenous concentration of 57Fe incorporated into RBCs after supplementation. In this way, to determine the mass of iron incorporated into erythrocytes, the unique prediction that must be made is the blood volume, estimate to reproduce the natural dilution of the tracer (57Fe) in the blood. This novel bioanalytical approach was applied for the measurements of iron incorporation and further iron absorption studies in humans, using a group of twelve healthy participants, that should be further evaluated for the assessment of other chemical elements that could be of health concerns and directly impact society.

A plasmonic MNAzyme signal amplification strategy for quantification of miRNA-4739 breast cancer biomarker.

A major challenge in the 21st century is the development of point-of-care diagnostic tools capable to detect and quantify disease biomarkers in a straightforward, affordable, sensitive, and specific manner. The remarkable plasmonic properties of gold nanoparticles (AuNPs) have promoted their use for development of simple methodologies for nucleic acid detection in combination with a variety of oligonucleotides amplification techniques. Here, assemblies of AuNPs with Multicomponent Nucleic Acid enzymes (MNAzymes) has been successfully used in the design of a highly sensitive and simple bioassay for rapid spectroscopic detection and quantification of miRNA-4739 in blood samples. The miRNA selected is a doxorubicin chemoresistant biomarker in breast cancer which overexpression promotes the proliferation, progression, and survival of cancer cells. In this work, two alternatives experimental designs, based on use of MNAzymes and AuNPs, have been optimized and applied for sensitive miRNA-4739 quantification: one based on a traditional direct measurement of wavelength shift and a second non-conventional simple approach based on isolation and measurement of free nanoparticles absorbance. Improvement in sensitivity and, higher measurement accuracy and precision were achieved with the second approach. The developed bioassay provides a detection limit as low as 7 pmolL-1 for miRNA-4739 quantification and performed satisfactory selectivity and well practical applicability by analysis of the miRNA-4739 in blood, demonstrating that the proposed strategy is a promising and suitable spectroscopic method for breast cancer diagnosis thought liquid biopsy of circulating tumoral cells.

Uncovering the Relationship between Selenium Status, Age, Health, and Dietary Habits: Insights from a Large Population Study including Nonagenarian Offspring from the MARK-AGE Project.

An inadequate selenium (Se) status can accelerate the aging process, increasing the vulnerability to age-related diseases. The study aimed to investigate plasma Se and Se species in a large population, including 2200 older adults from the general population (RASIG), 514 nonagenarian offspring (GO), and 293 GO Spouses (SGO). Plasma Se levels in women exhibit an inverted U-shaped pattern, increasing with age until the post-menopausal period and then declining. Conversely, men exhibit a linear decline in plasma Se levels with age. Subjects from Finland had the highest plasma Se values, while those from Poland had the lowest ones. Plasma Se was influenced by fish and vitamin consumption, but there were no significant differences between RASIG, GO, and SGO. Plasma Se was positively associated with albumin, HDL, total cholesterol, fibrinogen, and triglycerides and negatively associated with homocysteine. Fractionation analysis showed that Se distribution among plasma selenoproteins is affected by age, glucometabolic and inflammatory factors, and being GO or SGO. These findings show that sex-specific, nutritional, and inflammatory factors play a crucial role in the regulation of Se plasma levels throughout the aging process and that the shared environment of GO and SGO plays a role in their distinctive Se fractionation.

AF4-UV/VIS-MALS-ICPMS/MS for the characterization of the different nanoparticulated species present in oligonucleotide-gold nanoparticle conjugates.

In-depth characterization of functionalized nanomaterials is still a remaining challenge in nanobioanalytical chemistry. In this work, we propose the online coupling of Asymmetric Flow Field-Flow Fractionation (AF4) with UV/Vis, Multiangle Light Scattering (MALS) and Inductively Coupled Plasma-Tandem Mass Spectrometry (ICP-MS/MS) detectors to carry out, in less than 10 min and directly in the functionalization reaction mixture, the complete characterization of gold nanoparticles (AuNPs) functionalized with oligonucleotides and surface-modified with polyethylene glycol (PEG). AF4 separation provided full separation of the bioconjugates from the original AuNPs while P/Au and S/Au ICP-MS/MS ratios in the bioconjugate fractographic peaks could be used to compute the corresponding stoichiometries, oligonucleotide/AuNP and PEG/AuNPs. MALS detection clearly showed the coexistence of two distinct nanoparticulated populations in the bioconjugation mixture, which were demonstrated to be different not only in size but in functionality as well. The major bioconjugate population showed lower hydrodynamic ratios (18 nm) with higher and steadier oligonucleotides/AuNPs (92) and PEG/AuNPs (2350) stoichiometries, in comparison to the minor abundant population (54 nm, 51 and 1877, respectively). Moreover, the ratio between the absorbance signals measured at 520 nm and 650 nm reflects a lower AuNP aggregation in the major (10.5) than in the minor (4.5) population. Results obtained prove the benefits of a detailed characterization to find out if subsequent purification of functionalized AuNP-oligonucleotides is required to design more efficiently their final bioanalytical application.

Quantitative multiplexed analysis of MMP-11 and CD45 in metastatic breast cancer tissues by immunohistochemistry-assisted LA-ICP-MS.

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P = 0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P = 0.0077) and healthy tissues (P = 0.0087).

Near-Infrared Sensors for Onsite and Noninvasive Quantification of Macronutrients in Breast Milk.

Breast milk is an optimal food that covers all the nutritional needs of the newborn. It is a dynamic fluid whose composition varies with lactation period. The neonatal units of hospitals have human milk banks, a service that analyzes, stores, and distributes donated human milk. This milk is used to feed premature infants (born before 32 weeks of gestation or weighing less than 1500 g) whose mothers, for some reason, cannot feed them with their own milk. Here, we aimed to develop near-infrared spectroscopy (NIRS) measures for the analysis of breast milk. For this purpose, we used a portable NIRS instrument scanning in the range of 1396-2396 nm to collect the spectra of milk samples. Then, different chemometrics were calculated to develop 18 calibration models with and without using derivatives and the standard normal variate. Once the calibration models were developed, the best treatments were selected according to the correlation coefficients (r2) and prediction errors (SECVs). The best results for the assayed macronutrients were obtained when no pre-treatment was applied to the NIR spectra of fat (r2 = 0.841, SECV = 0.51), raw protein (r2 = 0.512, SECV = 0.21), and carbohydrates (r2 = 0.741, SECV = 1.35). SNV plus the first derivative was applied to obtain satisfactory results for energy (r2 = 0.830, SECV = 9.60) quantification. The interpretation of the obtained results showed the richness of the NIRS spectra; moreover, the presence of specific bands for fat provided excellent statistics in quantitative models. These results demonstrated the ability of portable NIRS sensors in a methodology developed for the quality control of macronutrients in breast milk.

Mesenchymal Stem Cells as a Cornerstone in a Galaxy of Intercellular Signals: Basis for a New Era of Medicine.

Around 40% of the population will suffer at some point in their life a disease involving tissue loss or an inflammatory or autoimmune process that cannot be satisfactorily controlled with current therapies. An alternative for these processes is represented by stem cells and, especially, mesenchymal stem cells (MSC). Numerous preclinical studies have shown MSC to have therapeutic effects in different clinical conditions, probably due to their mesodermal origin. Thereby, MSC appear to play a central role in the control of a galaxy of intercellular signals of anti-inflammatory, regenerative, angiogenic, anti-fibrotic, anti-oxidative stress effects of anti-apoptotic, anti-tumor, or anti-microbial type. This concept forces us to return to the origin of natural physiological processes as a starting point to understand the evolution of MSC therapy in the field of regenerative medicine. These biological effects, demonstrated in countless preclinical studies, justify their first clinical applications, and draw a horizon of new therapeutic strategies. However, several limitations of MSC as cell therapy are recognized, such as safety issues, handling difficulties for therapeutic purposes, and high economic cost. For these reasons, there is an ongoing tendency to consider the use of MSC-derived secretome products as a therapeutic tool, since they reproduce the effects of their parent cells. However, it will be necessary to resolve key aspects, such as the choice of the ideal type of MSC according to their origin for each therapeutic indication and the implementation of new standardized production strategies. Therefore, stem cell science based on an intelligently designed production of MSC and or their derivative products will be able to advance towards an innovative and more personalized medical biotechnology.

Total metal content and chemical speciation analysis of iron, copper, zinc and iodine in human breast milk using high-performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection.

The aim of this research was to quantify essential trace elements (iron, copper, zinc and iodine) and establish their speciation in human milk. Both the element and the species are important in new-born nutrition. Colostrum, and transitional and mature milks (25) were collected from 18 mothers of pre-term or full-term infants. Concentrations of the target elements were determined using ICP-MS. For speciation, HPLC coupled to ICP-MS was employed. Total contents of the micronutrients varied in mothers of pre-term (Fe = 0.997, Cu = 0.506, Zn = 4.15 and I = 0.458 mg L-1) and mothers of full-term (Fe = 0.733, Cu = 0.234, Zn = 2.91 and I = 0.255 mg L-1) infants. Fe, Cu and Zn were associated with biomolecules with high molecular mass compounds, such as immunoglobulins, albumin and lactoferrin whilst iodine was only found as iodide.

MMP-11 as a biomarker for metastatic breast cancer by immunohistochemical-assisted imaging mass spectrometry.

MMP-11 is a member of the matrix metalloproteinase family (MMPs) which are overexpressed in cancer cells, stromal cells and the adjacent microenvironment. The MMP protein family encompasses zinc-dependent endopeptidases that degrade the extracellular matrix (ECM), facilitating the breakdown of the basal membrane and matrix connective tissues. This function is believed to be important in cancer development and metastasis. This paper investigated a gold nanoparticle-based immunohistochemical assay to visualise the distribution of MMP-11 in human breast cancer tissues from eight patients with and without metastases by employing laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The expression of MMP-11 was increased and more heterogeneous in metastatic specimens compared to non-metastatic tumour samples. These findings demonstrate that imaging breast tumours by LA-ICP-MS may be a useful tool to aid the prognosis and treatment of breast cancer. As an example, samples of two patients are presented who were diagnosed with matching characteristics and grades of breast cancer. Although both patients had a similar prognosis and treatment, only one developed metastases.

Protective effect of selenium supplementation following oxidative stress mediated by glucose on retinal pigment epithelium.

There are many conditions that affect the retina. However, diabetic retinopathy (RD) as a complication of Diabetes Mellitus continues to be the leading cause of blindness in working people globally. Diabetic retinopathy is an ocular complication of diabetes that is caused by the deterioration of the blood vessels that supply the retina, which has the consequence that the vision deteriorates irreversibly. The retina, and specifically the retinal pigment epithelium (RPE) is the only neural tissue that is exposed directly and frequently to light, which favors the oxidation of lipids that become extremely toxic to the cells of the retina. The RPE is a natural barrier playing an important role in the absorption of light and reduction of light scatter within the eye. In addition, the retina is the tissue that proportionally consumes more oxygen, which generates a high production of reactive oxygen species (ROS). The retina is particularly sensitive to hyperglycemia and oxidative stress. The eye tissues are enriched in certain antioxidants in the form of metabolic enzymes or small molecules. Since selenium is essential for regulating the activity of the enzymes involved in protection against oxidative stress, providing selenium to the ocular tissues could be useful for the treatment of different ocular pathologies. Thus, the aim of this study is to investigate the potential efficacy of selenium in human RPE against glucose-induced oxidative stress and its implications for GPx activity. Chromatographic techniques based on HPLC-ICP-MS will be applied in combination with isotope pattern deconvolution (IPD) to study the effects of selenium supplementation and hyperglycemia in an in vitro model of RPE cells.

Selenium levels and Glutathione peroxidase activity in the plasma of patients with type II diabetes mellitus.

Selenium, an essential trace element, is involved in the complex system of defense against oxidative stress through selenium-dependent glutathione peroxidases (GPx) and other selenoproteins. Because of its antioxidant properties, selenium or its selenospecies at appropriate levels could hinder oxidative stress and so development of diabetes. In this vein, quantitative speciation of selenium in human plasma samples from healthy and diabetic patients (controlled and non-controlled) was carried out by affinity chromatography (AF) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) and isotope dilution analysis (IDA). Similarly, it is well known that patients with diabetes who exhibit poor control of blood glucose show a decreased total antioxidant activity. Thus, we evaluated the enzymatic activity of GPx in diabetic and healthy individuals, using the Paglia and Valentine enzymatic method, observing a significant difference (p<0.05) between the three groups of assayed patients (healthy (n=24): 0.61±0.11U/ml, controlled diabetic (n=38): 0.40±0.12U/ml and non-controlled diabetic patients (n=40): 0.32±0.09U/ml). Our results show that hyperglycemia induces oxidative stress in diabetic patients compared with healthy controls. What is more, glycation of GPx experiments demonstrated that it is the degree of glycation of the selenoenzyme (another species of the Se protein) what actually modulates its eventual activity against ROS in type II diabetes mellitus patients.

Quantitative selenium speciation by HPLC-ICP-MS(IDA) and simultaneous activity measurements in human vitreous humor.

A post-column isotope dilution analysis (IDA) methodology was applied to carry out quantitative speciation of selenium in human vitreous humor samples by size exclusion chromatography (SEC) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). Two main selenium species detected by SEC-ICP-MS were found to be associated to protein complexes. The expected molecular weights for both selenium-bound complexes were confirmed by MALDI-TOF(MS) and the results matched well with the theoretical mass of a GPx monomer (M, 22 kDa) and tetramer (T, 88 kDa). The quantification of the two detected selenium-bound complexes by post-column IDA showed that the total content of selenospecies in vitreous humor was approximately 3.2 ± 1.8 ppb Se. Moreover, in most of the analyzed vitreous humor samples, the majority of the selenium was associated to higher molecular weight GPx biomolecules. In an attempt to assess if the enzymatic activity was associated with a given selenium-bound GPx protein, the antioxidant enzyme activity was assayed for the two separated GPx species. Only for GPx (T) was a linear relationship between activity and total Se concentration found by ICP-MS.

Changes in clinical pain in fibromyalgia patients correlate with changes in brain activation in the cingulate cortex in a response inhibition task.

OBJECTIVE: The primary symptom of fibromyalgia is chronic, widespread pain; however, patients report additional symptoms including decreased concentration and memory. Performance-based deficits are seen mainly in tests of working memory and executive functioning. It has been hypothesized that pain interferes with cognitive performance; however, the neural correlates of this interference are still a matter of debate. In a previous, cross-sectional study, we reported that fibromyalgia patients (as compared with healthy controls) showed a decreased blood oxygen level dependent (BOLD) response related to response inhibition (in a simple Go/No-Go task) in the anterior/mid cingulate cortex, supplementary motor area, and right premotor cortex. METHODS: Here in this longitudinal study, neural activation elicited by response inhibition was assessed again in the same cohort of fibromyalgia patients and healthy controls using the same Go/No-Go paradigm. RESULTS: A decrease in percentage of body pain distribution was associated with an increase in BOLD signal in the anterior/mid cingulate cortex and the supplementary motor area, regions that have previously been shown to be "hyporeactive" in this cohort. CONCLUSIONS: Our results suggest that the clinical distribution of pain is associated with the BOLD response elicited by a cognitive task. The cingulate cortex and the supplementary motor area are critically involved in both the pain system as well as the response inhibition network. We hypothesize that increases in the spatial distribution of pain might engage greater neural resources, thereby reducing their availability for other networks. Our data also point to the potential for, at least partial, reversibility of these changes.

Nutritional iron supplementation studies based on enriched (57) Fe, added to milk in rats, and isotope pattern deconvolution-ICP-MS analysis.

Enriched stable iron isotopes in combination with isotope pattern deconvolution and ICP-MS have been used to study the absorption and bioavailability of iron from supplemented formula milk administrated to lactating rats. The use of two enriched stable isotope tracers, one as the metabolic tracer (here (57) Fe) and the other ((54) Fe) as quantitation tracer, is shown to provide quantitative data about endogenous and exogenous (supplemented) total Fe distribution in rat feces, urine, red blood cells (RBCs), serum, liver, and kidney. The proposed analytical methodology was validated using reference materials (serum, urine, and liver) spiked with both (54) Fe and (57) Fe. Quantitative information about iron absorption/bioavailability and/or metabolism can be obtained from the amounts of endogenous and exogenous iron found in the tissues and fluids analyzed, and about its kinetic after 2 weeks of iron supplementation. The obtained results are discussed in terms of iron exchanged and its half-life in lactating rats and the observed iron levels in serum, RBCs, liver, and kidney comparing nonsupplemented rats and maternal feed rats.

Executive function in chronic pain patients and healthy controls: different cortical activation during response inhibition in fibromyalgia.

UNLABELLED: The primary symptom of fibromyalgia (FM) is chronic, widespread pain; however, patients report additional symptoms including decreased concentration and memory. Performance-based deficits are seen mainly in tests of working memory and executive function. Neural correlates of executive function were investigated in 18 FM patients and 14 age-matched healthy controls during a simple Go/No-Go task (response inhibition) while they underwent functional magnetic resonance imaging (fMRI). Performance was not different between FM and healthy control, in either reaction time or accuracy. However, fMRI revealed that FM patients had lower activation in the right premotor cortex, supplementary motor area, midcingulate cortex, putamen and, after controlling for anxiety, in the right insular cortex and right inferior frontal gyrus. A hyperactivation in FM patients was seen in the right inferior temporal gyrus/fusiform gyrus. Despite the same reaction times and accuracy, FM patients show less brain activation in cortical structures in the inhibition network (specifically in areas involved in response selection/motor preparation) and the attention network along with increased activation in brain areas not normally part of the inhibition network. We hypothesize that response inhibition and pain perception may rely on partially overlapping networks, and that in chronic pain patients, resources taken up by pain processing may not be available for executive functioning tasks such as response inhibition. Compensatory cortical plasticity may be required to achieve performance on a par with control groups. PERSPECTIVE: Neural activation (fMRI) during response inhibition was measured in fibromyalgia patients and controls. FM patients show lower activation in the inhibition and attention networks and increased activation in other areas. Inhibition and pain perception may use overlapping networks: resources taken up by pain processing may be unavailable for other processes.

The emerging role of ICP-MS in proteomic analysis.

Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics. In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The "heteroatom (isotope)-tagged proteomics" concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a "hard" ion source, the ICP, to complement well-established "soft" ion sources for mass spectrometry to tackle present proteomic analysis.

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures.

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The 78Se-to-77Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.