Design and Development of a Bimodal Optical Instrument for Simultaneous Vibrational Spectroscopy Measurements

Vibrational spectroscopy techniques are widely used in analytical chemistry, physics and biology. The most prominent techniques are Raman and Fourier-transform infrared spectroscopy (FTIR). Combining both techniques delivers complementary information of the test sample. We present the design, construction, and calibration of a novel bimodal spectroscopy system featuring both Raman and infrared measurements simultaneously on the same sample without mutual interference. The optomechanical design provides a modular flexible system for solid and liquid samples and different configurations for Raman. As a novel feature, the Raman module can be operated off-axis for optical sectioning. The calibrated system demonstrates high sensitivity, precision, and resolution for simultaneous operation of both techniques and shows excellent calibration curves with coefficients of determination greater than 0.96. We demonstrate the ability to simultaneously measure Raman and infrared spectra of complex biological material using bovine serum albumin. The performance competes with commercial systems; moreover, it presents the additional advantage of simultaneously operating Raman and infrared techniques. To the best of our knowledge, it is the first demonstration of a combined Raman-infrared system that can analyze the same sample volume and obtain optically sectioned Raman signals. Additionally, quantitative comparison of confocality of backscattering micro-Raman and off-axis Raman was performed for the first time.     

Chronic Hyperkaliemia in Chronic Kidney Disease: An Old Concern with New Answers

Increasing potassium intake ameliorates blood pressure (BP) and cardiovascular (CV) prognoses in the general population; therefore the World Health Organization recommends a high-potassium diet (90–120 mEq/day). Hyperkalaemia is a rare condition in healthy individuals due to the ability of the kidneys to effectively excrete dietary potassium load in urine, while an increase in serum K⁺ is prevalent in patients with chronic kidney disease (CKD). Hyperkalaemia prevalence increases in more advanced CKD stages, and is associated with a poor prognosis. This scenario generates controversy on the correct nutritional approach to hyperkalaemia in CKD patients, considering the unproven link between potassium intake and serum K⁺ levels. Another concern is that drug-induced hyperkalaemia leads to the down-titration or withdrawal of renin-angiotensin system inhibitors (RASI) and mineralocorticoids receptors antagonists (MRA) in patients with CKD, depriving these patients of central therapeutic interventions aimed at delaying CKD progression and decreasing CV mortality. The new K⁺-binder drugs (Patiromer and Sodium-Zirconium Cyclosilicate) have proven to be adequate and safe therapeutic options to control serum K⁺ in CKD patients, enabling RASI and MRA therapy, and possibly, a more liberal intake of fruit and vegetables.     

S-Adenosylmethionine Inhibits Colorectal Cancer Cell Migration through Mirna-Mediated Targeting of Notch Signaling Pathway

Metastasis is a leading cause of mortality and poor prognosis in colorectal cancer (CRC). Thus, the identification of new compounds targeting cell migration represents a major clinical challenge. Recent findings evidenced a central role for dysregulated Notch in CRC and a correlation between Notch overexpression and tumor metastasis. MicroRNAs (miRNAs) have been reported to cross-talk with Notch for its regulation. Therefore, restoring underexpressed miRNAs targeting Notch could represent an encouraging therapeutic approach against CRC. In this context, S-adenosyl-L-methionine (AdoMet), the universal biological methyl donor, being able to modulate the expression of oncogenic miRNAs could act as a potential antimetastatic agent. Here, we showed that AdoMet upregulated the onco-suppressor miRNAs-34a/-34c/-449a and inhibited HCT-116 and Caco-2 CRC cell migration. This effect was associated with reduced expression of migration-/EMT-related protein markers. We also found that, in colorectal and triple-negative breast cancer cells, AdoMet inhibited the expression of Notch gene, which, by luciferase assay, resulted the direct target of miRNAs-34a/-34c/-449a. Gain- and loss-of-function experiments with miRNAs mimics and inhibitors demonstrated that AdoMet exerted its inhibitory effects by upregulating miRNAs-34a/-34c/-449a. Overall, these data highlighted AdoMet as a novel Notch inhibitor and suggested that the antimetastatic effects of AdoMet involve the miRNA-mediated targeting of Notch signaling pathway.     

Evaluation and Characterization of Post-Stroke Lung Damage in a Murine Model of Cerebral Ischemia

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics^® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham-controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage.     

Fighting the Huntington’s Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization,In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.     

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic β-Cell Model

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-βH1 β-cell line, the rat β-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as “unknowns”. Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a “new” diabetogenic agent. Our results indicate that the EndoC-βH1 cell line is a suitable human β-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.     

Polymerizable Skin Hydrogel for Full Thickness Wound Healing

The skin is the largest organ in the human body, comprising the main barrier against the environment. When the skin loses its integrity, it is critical to replace it to prevent water loss and the proliferation of opportunistic infections. For more than 40 years, tissue-engineered skin grafts have been based on the in vitro culture of keratinocytes over different scaffolds, requiring between 3 to 4 weeks of tissue culture before being used clinically. In this study, we describe the development of a polymerizable skin hydrogel consisting of keratinocytes and fibroblast entrapped within a fibrin scaffold. We histologically characterized the construct and evaluated its use on an in vivo wound healing model of skin damage. Our results indicate that the proposed methodology can be used to effectively regenerate skin wounds, avoiding the secondary in vitro culture steps and thus, shortening the time needed until transplantation in comparison with other bilayer skin models. This is achievable due to the instant polymerization of the keratinocytes and fibroblast combination that allows a direct application on the wound. We suggest that the polymerizable skin hydrogel is an inexpensive, easy and rapid treatment that could be transferred into clinical practice in order to improve the treatment of skin wounds.     

Formation of oxidising species and their role in the viscosity loss of cereal beta-glucan extracts

The aim of this study was to determine the role of the formation of radicals and hydrogen peroxide in the viscosity loss of beta-glucan extracts from oat and barley. Radicals derived from endogenous H₂O₂ were formed spontaneously in all extracts. The decomposition of H₂O₂ by catalase had little or no effect on the viscosity, and the radical formation detected and measured by electron spin resonance (ESR) spectroscopy did not predict the shelf life of crude beta-glucan extracts. The addition of FeSO₄ accelerated the radical formation and viscosity loss, revealing the sensitivity of the extract viscosity towards catalysts for oxidation. Using 50% cadoxen in the extracts slowed down the viscosity loss in all extracts but still led to a 10–70% decrease of the initial viscosities during 4 weeks of storage. The results suggest that the viscosity loss may be a result from multiple mechanisms including chemical reactions during storage as well as aggregation and conformational changes.