Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.     

Improving the Management of Endometrial Cancer Patients through the Use of Liquid Biopsy Analyses: A Case Report

Endometrial cancer (EC) is the 4th most common neoplasm of the female genital tract, with 15–20% of patients being of high risk of recurrence which leads to a significant decrease in patient survival. Current therapeutic options for patients with EC are poor, being the combined therapy of carboplatin and paclitaxel the standard of care, with limited efficacy. Therefore, new therapeutic options and better monitoring tools are needed to improve the management of the disease. In the current case report, we showcase the value of liquid biopsy analyses in a microsatellite instability EC patient with initially good prognosis that however underwent rapid progression disease within 6 months post-surgery; through the study of plasma cfDNA/ctDNA dynamics to assess the tumour evolution during treatment, as well as the study of the uterine aspirate as a valuable sample that captures the intra-tumour heterogeneity that allows a comprehensive genomic profiling of the disease to identify potential therapeutic options. Furthermore, preclinical models were generated at the time of tumour progression to assess the efficacy of the identified targeted therapies.     

Bacterial DNAemia in Alzheimer’s Disease and Mild Cognitive Impairment: Association with Cognitive Decline,Plasma BDNF Levels,and Inflammatory Response

Microbial dysbiosis (MD) provokes gut barrier alterations and bacterial translocation in the bloodstream. The increased blood bacterial DNA (BB-DNA) may promote peripheral- and neuro-inflammation, contributing to cognitive impairment. MD also influences brain-derived neurotrophic factor (BDNF) production, whose alterations contribute to the etiopathogenesis of Alzheimer’s disease (AD). The purpose of this study is to measure BB-DNA in healthy elderly controls (EC), and in patients with mild cognitive impairment (MCI) and AD to explore the effect on plasma BDNF levels (pBDNF), the inflammatory response, and the association with cognitive decline during a two-year follow-up. Baseline BB-DNA and pBDNF were significantly higher in MCI and AD than in EC. BB-DNA was positively correlated with pBDNF in AD, plasma Tumor necrosis factor-alpha (TNF-α), and Interleukin-10 (IL-10) levels in MCI. AD patients with BB-DNA values above the 50th percentile had lower baseline Mini-Mental State Examination (MMSE). After a two-year follow-up, AD patients with the highest BB-DNA tertile had a worse cognitive decline, while higher BB-DNA levels were associated with higher TNF-α and lower IL-10 in MCI. Our study demonstrates that, in early AD, the higher the BB-DNA levels, the higher the pBDNF levels, suggesting a defensive attempt; BB-DNA seems to play a role in the AD severity/progression; in MCI, higher BB-DNA may trigger an increased inflammatory response.     

Circulating Extracellular Vesicles Impair Mesenchymal Stromal Cell Differentiation Favoring Adipogenic Rather than Osteogenic Differentiation in Adolescents with Obesity

Excess body weight has been considered beneficial to bone health because of its anabolic effect on bone formation; however, this results in a poor quality bone structure. In this context, we evaluated the involvement of circulating extracellular vesicles in the impairment of the bone phenotype associated with obesity. Circulating extracellular vesicles were collected from the plasma of participants with normal weight, as well as overweight and obese participants, quantified by flow cytometry analysis and used to treat mesenchymal stromal cells and osteoblasts to assess their effect on cell differentiation and activity. Children with obesity had the highest amount of circulating extracellular vesicles compared to controls. The treatment of mesenchymal stromal cells with extracellular vesicles from obese participants led to an adipogenic differentiation in comparison to vesicles from controls. Mature osteoblasts treated with extracellular vesicles from obese participants showed a reduction in differentiation markers in comparison to controls. Children with obesity who regularly performed physical exercise had a lower circulating extracellular vesicle amount in comparison to those with a sedentary lifestyle. This pilot study demonstrates how the high amount of circulating extracellular vesicles in children with obesity affects the bone phenotype and that physical activity can partially rescue this phenotype.     

I2-Imidazoline Ligand CR4056 Improves Memory,Increases ApoE Expression and Reduces BBB Leakage in 5xFAD Mice

Recent evidence suggests that I2-imidazoline ligands have neuroprotective properties in animal models of neurodegeneration, such as Alzheimer’s disease (AD). We recently demonstrated that the I2-ligand BU224 reversed memory impairments in AD transgenic mice and this effect was not because of reductions in amyloid-β (Aβ) deposition. In this study, our aim was to determine the therapeutic potential of the powerful analgesic I2-imidazoline ligand CR4056 in the 5xFAD model of AD, since this ligand has been proven to be safely tolerated in humans. Sub-chronic oral administration of CR4056 (30 mg/kg for 10 days) led to an improvement in recognition memory in 6-month-old 5xFAD mice, but not in wild-type littermates, without affecting Aβ levels or deposition. Our results also revealed a change in the profile of microglia by CR4056, resulting in a suppression of pro-inflammatory activated microglia, but increased the density of astrocytes and the expression of ApoE, which is mainly produced by these glial cells. In addition, CR4056 restored fibrinogen extravasation, affecting the distribution of markers of astrocytic end feet in blood vessels. Therefore, these results suggest that CR4056 protects against Aβ-mediated neuroinflammation and vascular damage, and offers therapeutic potential at any stage of AD.     

Establishing In-House Cutoffs of CSF Alzheimer’s Disease Biomarkers for the AT(N) Stratification of the Alzheimer Center Barcelona Cohort

Background: Clinical diagnosis of Alzheimer’s disease (AD) increasingly incorporates CSF biomarkers. However, due to the intrinsic variability of the immunodetection techniques used to measure these biomarkers, establishing in-house cutoffs defining the positivity/negativity of CSF biomarkers is recommended. However, the cutoffs currently published are usually reported by using cross-sectional datasets, not providing evidence about its intrinsic prognostic value when applied to real-world memory clinic cases. Methods: We quantified CSF Aβ1-42, Aβ1-40, t-Tau, and p181Tau with standard INNOTEST^® ELISA and Lumipulse G^® chemiluminescence enzyme immunoassay (CLEIA) performed on the automated Lumipulse G600II. Determination of cutoffs included patients clinically diagnosed with probable Alzheimer’s disease (AD, n = 37) and subjective cognitive decline subjects (SCD, n = 45), cognitively stable for 3 years and with no evidence of brain amyloidosis in 18F-Florbetaben-labeled positron emission tomography (FBB-PET). To compare both methods, a subset of samples for Aβ1-42 (n = 519), t-Tau (n = 399), p181Tau (n = 77), and Aβ1-40 (n = 44) was analyzed. Kappa agreement of single biomarkers and Aβ1-42/Aβ1-40 was evaluated in an independent group of mild cognitive impairment (MCI) and dementia patients (n = 68). Next, established cutoffs were applied to a large real-world cohort of MCI subjects with follow-up data available (n = 647). Results: Cutoff values of Aβ1-42 and t-Tau were higher for CLEIA than for ELISA and similar for p181Tau. Spearman coefficients ranged between 0.81 for Aβ1-40 and 0.96 for p181TAU. Passing–Bablok analysis showed a systematic and proportional difference for all biomarkers but only systematic for Aβ1-40. Bland–Altman analysis showed an average difference between methods in favor of CLEIA. Kappa agreement for single biomarkers was good but lower for the Aβ1-42/Aβ1-40 ratio. Using the calculated cutoffs, we were able to stratify MCI subjects into four AT(N) categories. Kaplan–Meier analyses of AT(N) categories demonstrated gradual and differential dementia conversion rates (p = 9.815⁻²⁷). Multivariate Cox proportional hazard models corroborated these findings, demonstrating that the proposed AT(N) classifier has prognostic value. AT(N) categories are only modestly influenced by other known factors associated with disease progression. Conclusions: We established CLEIA and ELISA internal cutoffs to discriminate AD patients from amyloid-negative SCD individuals. The results obtained by both methods are not interchangeable but show good agreement. CLEIA is a good and faster alternative to manual ELISA for providing AT(N) classification of our patients. AT(N) categories have an impact on disease progression. AT(N) classifiers increase the certainty of the MCI prognosis, which can be instrumental in managing real-world MCI subjects.     

Application of Fast Gas Chromatography–Mass Spectrometry in Combination with the QuEChERS Method for the Determination of Pesticide Residues in Fruits and Vegetables

A fast gas chromatography–mass spectrometry method has been developed for multiresidue determination of up to 56 pesticides in fruits and vegetables in a chromatographic run time of <10 min, using a single quadrupole mass spectrometer operating in selected ion monitoring mode. The well-known acetate-buffering version of the QuEChERS method has been used for sample preparation. Programmable temperature vaporizer injection of 3 μL allowed reaching limits of detection between 0.15 and 15 μg/kg for most compounds in the sample matrices tested. The applicability of the method has been evaluated in apple, orange, carrot, and tomato. Recoveries at three fortification levels (0.01, 0.1 and 0.5 mg/kg) ranged from 70 to 120 % for most compounds, with relative standard deviations below 20 % in all cases. The developed method has been applied to fruit and vegetable samples from different Spanish provinces.