Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. ...Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.
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Adipose tissue (AT) dysfunctions are associated with the onset of insulin resistance (IR) and type 2 diabetes mellitus (T2DM). Targeting glucose-dependent insulinotropic peptide receptor (GIPR) is a ...valid option to increase the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists in T2DM treatment. Nevertheless, the therapeutic potential of targeting the GIP/GIPR axis and its effect on the AT are controversial. In this work, we explored the expression and regulation of GIPR in precursor cells and mature adipocytes, investigating if and how obesogenic stimuli and thiazolidinediones perturb GIPR expression. Using publicly available gene expression datasets, we assessed that, among white adipose tissue (WAT) cells, adipocytes express lower levels of GIPR compared to cells of mesothelial origin, pericytes, dendritic and NK/T cells. However, we report that GIPR levels markedly increase during the in vitro differentiation of both murine and human adipocytes, from 3T3-L1 and human mesenchymal precursor cells (MSCs), respectively. Notably, we demonstrated that thiazolidinediones – ie. synthetic PPARγ agonists widely used as anti-diabetic drugs and contained in the adipogenic mix – markedly induce GIPR expression. Moreover, using multiple in vitro systems, we assessed that thiazolidinediones induce GIPR in a PPARγ-independent manner. Our results support the hypothesis that PPARγ synthetic agonists may be used to increase GIPR levels in AT, potentially affecting in turn the targeting of GIP system in patients with metabolic dysfunctions. Furthermore, we demonstrate in vitro and in vivo that proinflammatory stimuli, and especially the TNFα, represses GIPR both in human and murine adipocytes, even though discordant results were obtained between human and murine cellular systems for other cytokines. Finally, we demonstrated that GIPR is negatively affected also by the excessive lipid engulfment. Overall, we report that obesogenic stimuli - ie. pro-inflammatory cytokines and the increased lipid accumulation – and PPARγ synthetic ligands oppositely modulate GIPR expression, possibly influencing the effectiveness of GIP agonists.
•Thiazolidinediones - synthetic PPARγ agonists - markedly induce GIPR expression in adipocytes and precursor cells.•Thiazolidinediones-mediated increase of GIPR is PPARγ independent.•Proinflammatory stimuli and excessive lipid engulfment strongly repress GIPR expression in mature adipocytes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We aimed to analyze the seasonal acclimatization process of Nelore and Canchim cattle raised on two production systems (non-shaded, NS, and integrated crop-livestock-forest, ICLF), based on the ...dynamics of the morphological and functional attributes of the hair coat and skin during winter and summer. The study was conducted in Brazil, in a low-altitude tropical climate region. A completely randomized 2 × 2 factorial design was adopted as follows: two production systems (NS and ICLF), two breeds (Nelore and Canchim) in a longitudinal structure, with measurements repeated over time through two stations (winter and summer). The experimental animals consisted of 32 Nelore (Bos indicus) and 32 Canchim (5/8 Bos taurus × 3/8 Bos indicus) bulls. The animals were equally distributed between two intensive rotational grazing systems. In both breeds, the hair coat was significantly thicker in winter but longer in summer, which increased epidermal protection. The Nelore bulls had shorter, wider, and thicker hairs, which are attributes that promote heat loss via conduction. The Canchim bulls showed significantly lower hair density and higher epithelium distance to sweat glands, which resulted in higher core temperature and respiratory rate. In turn, Nelore bulls had higher serum concentrations of triiodothyronine and lower serum concentrations of cortisol. However, Canchim bulls more frequently and intensely activated their thermoregulatory system and markedly adjusted their hair coat and hair features to reduce heat gain, especially in summer. Therefore, the anatomical plasticity and functional integumentary characteristics of Nelore and Canchim bulls reflect their acclimatization to tropical conditions.
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The synthesis and characterization of a novel water-compatible microsized material, based on fluorescent conjugated polymers (CPs), and its applicability for optical sensing of inorganic ions of ...environment interest (copper and cyanide) in water media is here described. Polyfluorene-based fluorescent CPs were synthesized and functionalized with imidazole moieties (selective recognition element) and a terminal double bond (covalently linked to an organic matrix) through a postfunctionalization strategy. Further, microspheres of the novel imidazole-functionalized fluorescent CPs, able to work in water media, were synthesized via a microemulsion and polymerization procedure. The synthesized imidazole-functionalized CP microspheres were then evaluated as fluorescence “turn-Off” sensing materials for Cu2+ detection in aqueous media. Analyte detection was based on the quenching effect of the Cu2+, selectively recognized by the imidazole group, on the polymer fluorescence emission. The developed optosensor exhibits a detection limit of 1 μg/L for the determination of Cu2+ in water with a reproducibility of 4%. The synthesized microsized material was also evaluated for the “turn-on” optosensing of cyanide in water, measuring the recovery of the emission signal from the CP that has been previously deactivated by the presence of quencher species. The “turn-On” optosensor allows the selective determination of free cyanide in aqueous solution with high sensitivity (detection limit of 18 μg/L), obtaining a reproducibility of 2.9%. A high sample throughput (between 7 and 12 samples per hour) was achieved in both cases. Analytical applicability of the fluorescent CP microsphere materials has been successfully demonstrated by tap and mineral water analysis.
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The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the ...pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers,
and
genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor) samples revealed that the transcription of
,
, and
is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.
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Continuous illumination induces the degeneration of photoreceptors. This animal model of light-induced retinal degeneration resembles many characteristics of human degenerative diseases of the outer ...retina, such as age-related macular degeneration. This work aimed to evaluate the potential neuroprotective effect of the modulation of adenosine A2A receptor in the model of light-induced retinal degeneration. Sprague-Dawley rats were intravitreally injected in the right eye with either CGS 21680, an adenosine A2A receptor agonist, or SCH 58261, an adenosine A2A receptor antagonist. Contralateral eyes were injected with respective vehicles as control. Then, rats were subjected to continuous illumination (12,000 lux) for 24 h. Retinas were processed by glial fibrillary acidic protein (GFAP) immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, Western blotting (WB), and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Another group of rats was subjected to functional studies by electroretinography. Animals treated with CGS21680 showed a significant increase of apoptotic nuclei in the outer nuclear layer and a significant increase of GFAP immunoreactive area of the retinas but did not alter WB nor electroretinography results. qRT-PCR showed that CGS 21680 significantly increased the expression of interleukin-1β. On the opposite, SCH 58261 significantly decreased apoptotic nuclei in the outer nuclear layer and GFAP immunoreactive area of the retinas. It also significantly decreased GFAP and activated caspase-3 levels as measured by WB and preserved retinal function, as treated eyes showed significantly greater amplitudes of a- and b-waves and oscillatory potentials. qRT-PCR revealed that SCH 58261 significantly decreased the expression of tumor necrosis factor-α. These results show that the blockade of the A2A receptor before the start of the pathogenic process is neuroprotective, as it prevents light-induced retinal damage. The use of A2A receptor antagonists deserves to be evaluated in retinal degenerative diseases.
Previously, we showed that 1‐nitro‐2‐phenylethene, a nitrostyrene derivative of 1‐nitro‐2‐phenylethane, induced vasorelaxant effects in rat aorta preparations. Here, we studied mechanisms underlying ...the vasorelaxant effects of its structural analog, trans‐4‐chloro‐β‐nitrostyrene (T4CN), in rat aortic rings. Increasing concentrations of T4CN (0.54‐544.69 µm) fully and similarly relaxed contractions induced by phenylephrine (PHE, 1 µm) or KCl (60 mm) in endothelium‐intact aortic rings with IC50 values of 66.74 59.66–89.04 and 79.41 39.92–158.01 µm, respectively. In both electromechanical and pharmacomechanical couplings, the vasorelaxant effects of T4CN remained unaltered by endothelium removal, as evidenced by the IC50 values (108.35 56.49–207.78 and 65.92 39.72–109.40 µm, respectively). Pretreatment of endothelium‐intact preparations with L‐NAME, ODQ, glibenclamide, or TEA did not change the vasorelaxant effect of T4CN. Under Ca2+‐free conditions, T4CN significantly reduced the phasic contractions induced by caffeine or PHE, as well as the contractions due to exogenous CaCl2 in aortic preparations stimulated with PHE (in the presence of verapamil). These results suggest that in rat aortic rings, T4CN induced vasorelaxation independently from the activation of soluble guanylate cyclase/cGMP pathway, an effect that may be related to the electrophilicity of the substituted chloro‐nitrostyrene. This vasorelaxation seems to involve inhibition of both calcium influx from the extracellular milieu and calcium mobilization from intracellular stores mediated by IP3 receptors and by ryanodine‐sensitive Ca2+ channels.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn ...must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of 34S and a generic S-containing internal standard spiked to the sample provides full compound independent detector response and thus protein quantification without the need for specific standards. Quantitative recoveries, using a chromatographic core–shell C4 column for the various protein species assayed were obtained (96–100%). Thus, the proposed strategy enables the accurate quantification of proteins even if no specific standards are available for them. In addition, to the best of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of intact proteins by direct measurement of sulfur with ICPMS (358 fmol) and protein (ranging from 7 to 15 fmol depending on the assayed protein). The quantitative results for individual and simple mixtures of model proteins were statistically indistinguishable from the manufacturer’s values. Finally, the suitability of the strategy for real sample analysis (including quantitative protein recovery from the column) was illustrated for the individual absolute quantification of the proteins and whole protein content in a venom sample. Parallel capLC–ESI-QTOF analysis was employed to identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic peak into individual protein mass.
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Context:
The fourth International Workshop on the Management of Asymptomatic Primary Hyperparathyroidism (PHPT) has recently suggested that skeletal and renal imaging be routinely conducted. So far, ...no study has systematically assessed this issue.
Objective:
The objective was to evaluate the prevalence of kidney stones (KS) and vertebral fractures (VFs) in a cohort of patients with PHPT utilizing noninvasive imaging technology.
Design:
This was a prospective study evaluating patients consecutively diagnosed with PHPT in a single center over a 5-year period (2009–2013).
Setting:
The setting was a referral center.
Patients:
There were a total of 140 patients with PHPT (127 women 18 premenopausal and 109 postmenopausal and 13 men; mean age, 63.2 ± 11 y).
Main Outcomes Measures:
Main outcome measures were the prevalence of KS by abdominal ultrasound, osteoporosis by dual-energy x-ray absorptiometry (DXA) (lumbar spine, femoral neck, total hip, and distal 1/3 radius), and VFs by vertebral spine x-ray, with attention to those categorized as symptomatic or asymptomatic.
Results:
Fifty-five percent of all subjects had KS by ultrasound, 62.9% had osteoporosis by T-score at any site, and 35.1% had VFs by x-ray. There was no difference in the incidence of VFs and densitometric osteoporosis between symptomatic and asymptomatic patients (VFs, 34.4 vs 34.7%; osteoporosis by DXA, 59.4 vs 65.8%), whereas more KS were detected in symptomatic (78%) than asymptomatic (35.5%). Twenty-two percent of patients classified as asymptomatic at baseline without osteoporosis by DXA were found to have KS and/or VFs.
Conclusions:
Nephrolithiasis and VFs are common in asymptomatic subjects with PHPT. The results provide evidence in support of recent recommendations that a more proactive approach be taken to detect silent bone and stone disease in asymptomatic PHPT.
Energy depletion caused by ischemic brain insults may result in persistent neuronal depolarization accompanied by hyper-stimulation of ionotropic glutamate receptors and excitotoxic phenomena, ...possibly leading to cell death. The use of glutamate receptor antagonists, such as the AMPARs antagonist Perampanel (PER), might be a pharmacological approach to counteract the excessive over-activation of glutamate receptors providing neuroprotective effects. Using electrophysiological and molecular analyses, we investigated the effect of PER against in vitro ischemia obtained by oxygen and glucose deprivation (OGD) in rat slices of two brain structures particularly sensitive to ischemic insults, the nucleus striatum and the hippocampus. We found that in these regions PER was able to avoid the OGD-induced neuronal suffering, at low doses not reducing basal excitatory synaptic transmission and not altering long-term potentiation (LTP) induction. Furthermore, in both the analysed regions, PER blocked a pathological form of LTP, namely ischemic LTP (iLTP). Finally, we hypothesized that the protective effect of PER against OGD was due to its capability to normalize the altered synaptic localization and function of AMPAR subunits, occuring after an ischemic insult. Taken together these findings support the idea that PER is a drug potentially effective to counteract ischemic damage.
•Low doses of Perampanel did not alter synaptic plasticity.•Perampanel preserves neuronal function during oxygen and glucose deprivation.•Oxygen and glucose deprivation induces a modification in AMPARs subunit composition.•Perampanel normalizes AMPARs subunit composition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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