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MicroRNAs (miRNAs) are promising drug targets for obesity and metabolic disorders. Recently, miRNA mimics are providing a unique mechanism of action that guides the process for drug development and sets out the context of their therapeutic application. miRNA (miR)-21 expression in white adipose tissue (WAT) has been associated with obesity. We aimed to analyze miR-21 expression levels in relation to diabetes and obesity to determine the effect that miR-21 mimic has on processes involved in WAT functionality, to dissect the underlying molecular mechanisms, and to study the potential therapeutic application of the miR-21 mimic against obesity. We found higher miR-21 levels in WAT from non-diabetic obese compared to normoweight humans and mice. Moreover, in 3T3-L1 adipocytes, miR-21 mimic affect genes involved in WAT functionality regulation and significantly increase the expression of genes involved in browning and thermogenesis. Interestingly, in vivo treatment with the miR-21 mimic blocked weight gain induced by a high-fat diet in obese mice, without modifying food intake or physical activity. This was associated with metabolic enhancement, WAT browning, and brown adipose tissue (AT) thermogenic programming through vascular endothelial growth factor A (VEGF-A), p53, and transforming growth factor β1 (TGF-β1) signaling pathways. Our findings suggest that miR-21 mimic-based therapy may provide a new opportunity to therapeutically manage obesity and consequently, its associated alterations., Graphical abstract, miR-21 mimic could serve as a potential therapeutic tool to control obesity by delaying energy storage and promoting energy expenditure in adipose tissue.

Electrogenerated chemiluminescence (ECL) efficiencies, redox potentials, photoluminescent (PL) (quenching and coupling) effects, and AFM images for the [Ru(bpy)3]2+/Au@tiopronin system were determined in aqueous solutions of the gold nanoparticles (NPs) at pH 7.0. The most remarkable finding was that ECL measurements can display the nanoparticle-induced resonance energy transfer (NP-RET) effect. Its effectiveness was quantified through a coefficient, K(NP-RET)ECL, which measures how much an ECL reaction has been enhanced. Moreover, the NP-RET effect was also checked using PL measurements, in such a way that a coefficient, K(NP-RET)PL, was determined; both constants, K(NP-RET)ECL and K(NP-RET)PL being in close agreement. It is important to highlight the fact that the NP-RET effect is only displayed in diluted solutions in which there is no NPs self-aggregation. The existence of the NPs self-aggregation behavior is revealed through AFM measurements.

Simple Summary Power Doppler is a non-invasive imaging technique that allows complete monitoring of the ovary changes in cycling mares. We use Power Doppler to investigate differences in follicular diameter and corpus luteum area as well as in follicular and corpus luteum blood flows between young and mature Spanish Purebred mares. Young mares had higher follicular and corpus luteum blood flows as well as higher blood progesterone levels. Moreover, we found that blood progesterone levels could be predicted in both groups from corpus luteum blood flow with moderate precision and accuracy. These results support the usefulness of Power Doppler to monitor ovarian hemodynamics and the suitability of corpus luteum blood flow to estimate blood progesterone levels in cycling mares. Abstract In equine reproduction, accurate and timely detection of the moment of ovulation is of great importance. Power Doppler ultrasound technology is a non-invasive method that enables to assess the morpho-echogenic features and blood flow changes during the estral cycle in mares. The objective of the present study was to evaluate the influence of age on ultrasonographic parameters (follicular diameter, follicular blood flow—FBF, corpus luteum (CL) area and corpus luteum blood flow—CLBF) and blood plasma progesterone concentrations in cycling Spanish Purebred mares (15 less than 8 years old and 15 equal o higher than 8 years old). The ultrasound images obtained were analyzed with the Image Colour Summarizer software, which allows the quantification of the pixels of each image. Young mares had significantly higher FBF, CLBF and plasma progesterone levels. Moreover, linear regression analysis showed that blood progesterone levels could be predicted in both groups from CLBF with moderate precision and accuracy. In conclusion, Power Doppler was useful to assess ovarian hemodynamics. Our results support that age is a factor that significantly influences FBF and CLBF as well as blood progesterone concentration in mares. More studies would be needed to develop high precision and accuracy predictive models of blood progesterone concentration from CLBF measured by Power Doppler.

Impaired adipose tissue (AT) lipid handling and inflammation is associated with obesity-related metabolic diseases. Circulating lipopolysaccharides (LPSs) from gut microbiota (metabolic endotoxemia), proposed as a triggering factor for the low-grade inflammation in obesity, might also be responsible for AT dysfunction. Nevertheless, this hypothesis has not been explored in human obesity. To analyze the relationship between metabolic endotoxemia and AT markers for lipogenesis, lipid handling, and inflammation in human obesity, 33 patients with obesity scheduled for surgery were recruited and classified according to their LPS levels. Visceral and subcutaneous AT gene and protein expression were analyzed and adipocyte and AT in vitro assays performed. Subjects with obesity with a high degree of metabolic endotoxemia had lower expression of key genes for AT function and lipogenesis ( SREBP1, FABP4, FASN, and LEP) but higher expression of inflammatory genes in visceral and subcutaneous AT than subjects with low LPS levels. In vitro experiments corroborated that LPS are responsible for adipocyte and AT inflammation and downregulation of PPARG, SCD, FABP4, and LEP expression and LEP secretion. Thus, metabolic endotoxemia influences AT physiology in human obesity by decreasing the expression of factors involved in AT lipid handling and function as well as by increasing inflammation.

The design and preparation of novel nanocarriers to transport cancer drugs for chemotherapy purposes is an important line of research in the medical field. A new 5-fluorouracil (5-Fu) transporter was designed based on the use of two new biocompatible gold nanosystems: (i) a gold nanoparticle precursor, Au@16-Ph-16, stabilized with the positively charged gemini surfactant 16- Ph-16, and (ii) the compacted nanocomplexes formed by the precursor and DNA/5-Fu complexes, Au@16-Ph-16/DNA-5-Fu. The physicochemical properties of the obtained nanosystems were studied by using UV-visible spectroscopy, TEM, dynamic light scattering, and zeta potential techniques. Method tuning also requires the use of circular dichroism, atomic force microscopy, and fluorescence spectroscopy techniques for the prior selection of the optimal relative Au@16-Ph-16 and DNA concentrations (R = CAu@16-Ph-16/CDNA), biopolymer compaction/decompaction, and 5-Fu release from the DNA/5-Fu complex. TEM experiments revealed the effective internalization of the both precursor and Au@16-Ph-16/DNA-5-Fu-compacted nanosystems into the cells. Moreover, cytotoxicity assays and internalization experiments using TEM and confocal microscopy showed that the new strategy for 5-Fu administration enhanced efficacy, biocompatibility and selectivity against lung cancer cells. The differential uptake among different formulations is discussed in terms of the physicochemical properties of the nanosystems. Universidad de Sevilla 2019/00000570, 2020/00001068, 2010/00000762, PP2016-5937, 2020/00001073 Junta de Andalucía 1804032996/2017, 2018/00000810, 2019/FQM-386

microRNAs are promising drug targets in obesity and metabolic disorders. miR-21 expression is upregulated in obese white adipose tissue (WAT); however, its physiological role in WAT has not been fully explored. We aimed to dissect the underlying molecular mechanisms of miR-21 in treating obesity, diabetes, and insulin resistance. We demonstrated, in human and mice, that elevated miR-21 expression is associated with metabolically healthy obesity. miR-21 mimic affected the expression of genes associated with adipogenesis, thermogenesis, and browning in 3T3-L1 adipocytes. In addition, it blocked high fat diet-induced weight gain in obese mice, without modifying food intake or physical activity. This was associated with metabolic enhancements, WAT browning and thermogenic programming, and brown AT induction through VEGF-A, p53, and TGFβ1 signaling pathways. Our findings add a novel role of miR-21 in the regulation of obesity and a potential therapy for both obesity and T2D without altering caloric intake and physical activities.

The efficiency of the high-energy electrogenerated chemiluminescence (ECL) tris(2,2′-bipyridine)ruthenium(II)/oxalate reaction was determined in the presence of citrate-coated gold nanoparticle (AuNPs) aqueous solutions. The ECL efficiency (ΦECL) for this reaction is enhanced by the addition of increasing amounts of AuNPs, followed by a sharp decrease when [AuNPs] is higher than 1.6 × 10–8 M. To explain this ΦECL/[AuNPs] trend, photoluminescent (PL) quenching, transmission electronic microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS) measurements were also performed as well as redox potentials for the [Ru(bpy)3]3+/2+ couple. Two facts were noteworthy: First, there is an increase in ΦECL, which accompanies an energy transfer effect by coupling between the Ru(II) complex ECL emission band and the AuNPs surface plasmon resonance (SPR)-induced band. This coupling, which we have called the ECL-SPR effect, enabled the quantifying of a coefficient (KECL-SPR, ECL-SPR effectiveness) for the improved ...

The interaction between calf thymus DNA and the gemini surfactants N,N'-[α,ω-phenylenebis(methylene)bis [N,N'-dimethyl-N-(1-hexadecyl)]-ammonium dibromide], p-16-Ph-16 (α = 1, ω = 3) and m-16-Ph-16 (α = 1, ω = 2), has been investigated via circular dichroism, fluorescence and UV-vis spectroscopy, zeta potential, dynamic light scattering, and AFM microscopy. Measurements were carried out in aqueous media at different molar ratios, R = (C16-Ph-16)/CDNA and C16-Ph-16 always below the critical micellar concentration (CMC) of the surfactant. Under these conditions, DNA undergoes two reversible conformational changes, compaction and decompaction, due to interaction with the surfactant molecules at low and high molar ratios, respectively. The extent of such conformational changes is correlated with both the degree of surfactant partial intercalation, and the size and charge of the surfactant aggregates formed, in each case. Comparison of the results shows that the para-form of the surfactant intercalates into the DNA to a major extent; therefore, the compaction/decompaction processes are more effective. Among these, the structure of the resulting 16-Ph-16/DNA decompacted complex is worthy of note. For the first time it can be demonstrated that the partial intercalation of the 16-Ph-16 gemini surfactants induces the formation of triplex DNA-like structures at a high R ratio.

The effect of the addition of low concentrations of an inner electrolyte on ds-DNA CT-DNA (calf thymus DNA) and ss-DNA conformational changes induced by small N-(2-mercaptopropionyl)glycine gold nanoparticles (AuNPs) is here studied in detail by using different spectroscopic and structural techniques. The high affinity of ss-DNA to AuNPs compared with ds-DNA is easily demonstrated by the results of competitive binding with SYBR Green I (SG). Additionally, it is proven that at 25.0 °C, AuNPs/ds-DNA and AuNPs/ss-DNA complexes undergo a transition from extended-coil to more compact structures when the AuNPs concentration (CAuNPs) is increased, which for the ds-DNA system is accompanied by partial denaturation. Particularly, for the AuNPs/ss-DNA system all of these techniques confirm that at a high CAuNPs, the compaction process is followed by a discrete transition to aggregation and an increase in structure size. A thorough analysis of the conformational changes described indicates that these processes are larger in low salt concentration and at high temperature. However, the most striking feature of this work is the abnormal melting temperature profiles (Tm) registered at high R = CAuNPs/CDNA ratios, which are remarkable and of interest for chemical sensing. At a suitable R ratio, which varies depending on CNaCl, a complex melting profile for the AuNPs/ds-DNA system was registered with two characteristic transitions: Tm,1 = 65.0 °C and Tm,2 = 95.0 °C. The highly sensitive atomic force microscopy technique performed at 25.0 °C and 65.0 °C also showed a different behaviour in both ss- and AuNPs/ds-DNA systems, which explains the characteristic melting curves. Specifically for the AuNPs/ss-DNA system, AFM at 25.0 °C revealed the formation of large-sized aggregates formed by AuNPs/ss-DNA compact structures linked by AuNPs. However, when both AuNPs/ds-DNA and AuNPs/ss-DNA complexes were incubated at 65.0 °C, the formation of highly stable ordered structures was always visualized at high R. Therefore, this shows that some key parameters for effective control of the formation of DNA/RNA-linked particles are: the selection of an optimal temperature below the ds-DNA melting point, an appropriate CAuNPs, and the addition of low CNaCl. The optimization of these parameters for each AuNPs/DNA system could improve biological sensing and DNA/RNA delivery.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western-blotting, and also by enzyme-linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age-dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.

We report a practical, economical and sensitive method for DNA and lysozyme biomolecule detection based on previous aggregation of an anionic citrate gold nanoparticle, Au@citrate, induced by N,N’-bis(dimethyldodecyl)-α,ω-alkanediammonium dibromide series (m-s-m, m = 12, s = 3 and 6) gemini surfactants. Starting from Au@citrate/12−s−12 aggregated complexes, disaggregation processes were induced, effectively and immediately, by increasing biopolymer concentrations. This phenomenon that changes the optical properties of the systems was originated by the strong, kinetically rapid interactions of Au@citrate with the biopolymer, favoring the formation of different highly stabilized Au@citrate/biopolymer/12−s−12 complexes, which particular structures were visualized via the AFM technique. In all cases, the order of addition of the reagents was demonstrated to be crucial for the success of the reaction, and therefore effective biomolecule detection. The new approach highlights a way to avoid problems derived from the time lapse observed in previously reported sensors based on gold nanoparticle’ aggregation phenomena, with a limit of detection in the nanomolar range. The results showed a better linear response in sensors prepared from 12−6−12 surfactant, in line with both the greater difference observed between the equilibrium binding constants for 12−6−12/biopolymer and Au@citrate/biopolymer interactions, and the significant interparticle distance among Au@citrate nanoparticles through the aggregates.

MicroRNAs (miRNAs) provide a unique mechanism of gene regulation and play a key role in different pathologies ranging from metabolic diseases to cancer. Thus, they are attractive candidates as therapeutic targets in the clinic. However, delivering miRNAs to specific tissues remains challenging, due to their inherent instability and their low diffusion into tissues, as well as a lack of effective means of delivering them to the cytosols of the specific cells. To overcome these challenges, we constructed a delivery vehicle based on novel gold nanosystems having gemini surfactants which offer a high capacity to induce the miRNAs compression, high stability and cell-entry capability. The synthesis of gold nanoparticles with a positive surface charge, Au@16-Ph-16/miR-21 and Au@16–3-16/miR-21, was carried out in a three-step process. In this method, the hydrogen tetrachloroaurate, sodium tetrahydroborate and 16-Ph-16 or 16–3-16 compounds were used as gold precursor, reducing agent and stabilizers, respectively. The nanoparticles obtained were then covered with miR-21 polymer and were characterized by UV–visible spectroscopy, transmission electron microscopy, atomic force microscopy, dynamic light scattering and zeta potential to measure size and charge distribution. Finally, toxicity was assessed in vivo in mice. Moreover, coherent anti-Stokes Raman spectroscopy and histochemistry analyses showed that Au@16-Ph-16/miR-21 and Au@16–3-16/miR-21 exhibited no toxicity, antimicrobial and biocompatible activities. These nanosystems can be a valuable alternative in therapies associated with diseases where miRNAs play a regulatory role that may be decisive in the improvement or cure of patients. All these characteristics could make of these nanosystems the best potential therapeutic molecules.

The equilibria and kinetics aspects of the binding of small gold nanoparticles, AuNPs, stabilized with tiopronin to DNA in B and C conformation (B-DNA and C-DNA), has been investigated in ethanol/water mixtures using different techniques. Two modes of binding are displayed: groove binding and partial intercalation, depending on the ethanol content, [EtOH], and the molar ratio, R = CAuNPs/CDNA. Two reaction mechanisms are proposed for AuNPs/DNA interaction in each polymer conformation, and the reaction parameters are evaluated. For lower ethanol levels, ([EtOH] up to 30%), when DNA is in the B form, the simplest mechanism according to the kinetic and thermodynamic results proved to be a three-step series mechanism reaction scheme which evolves in the formation of the groove complex. In this context, solvent hydration as well as the solvent effective viscosity are the main factors that influence kinetics. In contrast, for high ethanol levels, when DNA is in a C-like conformation, the mechanism is more complex involving three parallel reactions, in which AuNPs self-aggregation plays a key role in the switch from partial intercalation to groove binding. On the whole, it is evident that AuNPs aggregation and the DNA conformation are two key factors that must be taken into account in order to control the mechanism of AuNPs/DNA interaction.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of the Fmr1 gene product, fragile X mental retardation protein. Here we analyze the immunohistochemical expression of calcium-binding proteins in the dorsal thalamus of Fmr1 knockout mice of both sexes and compare it with that of wildtype littermates. The spatial distribution pattern of calbindin-immunoreactive cells in the dorsal thalamus was similar in wildtype and knockout mice but there was a notable reduction in calbindin-immunoreactive cells in midline/intralaminar/posterior dorsal thalamic nuclei of male Fmr1 knockout mice. We counted the number of calbindin-immunoreactive cells in 18 distinct nuclei of the dorsal thalamus. Knockout male mice showed a significant reduction in calbindin-immunoreactive cells (range: 36-67% lower), whereas female knockout mice did not show significant differences (in any dorsal thalamic nucleus) when compared with their wildtype littermates. No variation in the calretinin expression pattern was observed throughout the dorsal thalamus. The number of calretinin-immunoreactive cells was similar for all experimental groups as well. Parvalbumin immunoreactivity was restricted to fibers and neuropil in the analyzed dorsal thalamic nuclei, and presented no differences between genotypes. Midline/intralaminar/posterior dorsal thalamic nuclei are involved in forebrain circuits related to memory, nociception, social fear, and auditory sensory integration; therefore, we suggest that downregulation of calbindin protein expression in the dorsal thalamus of male knockout mice should be taken into account when analyzing behavioral studies in the mouse model of FXS.

This work investigates the nitrergic and cholinergic systems in the brain and spinal cord of the goldfish (Carassius auratus). We studied the immunohistochemical localization of antibodies against the neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) by bright-field and confocal microscopy. Nitrergic and cholinergic cells were segregated within the telencephalon, in both dorsal and ventral areas, and co-distributed in some nuclei of the diencephalon, mesencephalon, rhombencephalon, and spinal cord. Double-labeling experiments revealed nNOS/ChAT-positive cells in (1) the diencephalon: the preoptic and suprachiasmatic nuclei, the habenula, the dorsal thalamus, and the nucleus of the medial longitudinal fasciculus; (2) the mesencephalon: the optic tectum, the mesencephalic portion of the trigeminal nucleus, the oculomotor and trochlear nuclei, and the Edinger-Westphal nucleus; and (3) the rhombencephalon: the secondary gustatory nucleus, the nucleus isthmi, the lateral lemniscus nucleus, the cerebellum, the reticular formation, different nuclei of the octaval column, the motor zone of the vagal lobe, and the trigeminal, facial, abducens, glosso-pharyngeal, vagal, and hypobranchial motor nuclei. Double-labeled cells were also observed in the spinal motor column. The percentage of double-labeled cells was different in each studied nucleus, indicating a selective distribution pattern. Because double-labeled cells were more abundant in those nuclei involved with sensory and motor physiological processes, we suggest the involvement of both nitric oxide and acetylcholine in these neural functions in fish.

The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.

Parkinson’s disease (PD) is not only characterized by motor disturbances but also, by cognitive, sensory, psychiatric and autonomic dysfunction. It has been proposed that some of these symptoms might be related to the widespread pathology of α-synuclein (α-syn) aggregation in different nuclei of the central and peripheral nervous system. However, the pathogenic formation of α-syn aggregates in different brain areas of PD patients is poorly understood. Most experimental models of PD are valuable to assess specific aspects of its pathogenesis, such as toxin-induced dopaminergic neurodegeneration. However, new models are required that reflect the widespread and progressive formation of α-syn aggregates in different brain areas. Such α-syn aggregation is induced in only a few animal models, for example perikaryon inclusions are found in rats administered rotenone, aggregates with a neuritic morphology develop in mice overexpressing either mutated or wild-type α-syn, and in Smad3 deficient mice, aggregates form extensively in the perikaryon and neurites of specific brain nuclei. In this review we focus on α-syn aggregation in the human disorder, its genetics and the availability of experimental models. Indeed, evidences show that dopamine (DA) metabolism may be related to α-syn and its conformational plasticity, suggesting an interesting link between the two pathological hallmarks of PD: dopaminergic neurodegeneration and Lewy body (LB) formation.