VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

Accuracy and Survival Outcomes after National Implementation of Sentinel Lymph Node Biopsy in Early Stage Endometrial Cancer.

BACKGROUND: Sentinel lymph node (SLN) biopsy has recently been accepted to evaluate nodal status in endometrial cancer at early stage, which is key to tailoring adjuvant treatments. Our aim was to evaluate the national implementation of SLN biopsy in terms of accuracy to detect nodal disease in a clinical setting and oncologic outcomes according to the volume of nodal disease. PATIENTS AND METHODS: A total of 29 Spanish centers participated in this retrospective, multicenter registry including patients with endometrial adenocarcinoma at preoperative early stage who had undergone SLN biopsy between 2015 and 2021. Each center collected data regarding demographic, clinical, histologic, therapeutic, and survival characteristics. RESULTS: A total of 892 patients were enrolled. After the surgery, 12.9% were suprastaged to FIGO 2009 stages III-IV and 108 patients (12.1%) had nodal involvement: 54.6% macrometastasis, 22.2% micrometastases, and 23.1% isolated tumor cells (ITC). Sensitivity of SLN biopsy was 93.7% and false negative rate was 6.2%. After a median follow up of 1.81 years, overall surivial and disease-free survival were significantly lower in patients who had macrometastases when compared with patients with negative nodes, micrometastases or ITC. CONCLUSIONS: In our nationwide cohort we obtained high sensitivity of SLN biopsy to detect nodal disease. The oncologic outcomes of patients with negative nodes and low-volume disease were similar after tailoring adjuvant treatments. In total, 22% of patients with macrometastasis and 50% of patients with micrometastasis were at low risk of nodal metastasis according to their preoperative risk factors, revealing the importance of SLN biopsy in the surgical management of patients with early stage EC.

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Intravascular laser irradiation of blood (ILIB) used to treat lung diseases: a short critical review.

Intravascular laser irradiation of blood (ILIB) was developed to treat cardiovascular diseases due to its rheological effects. In its original form, ILIB was applied by an intravenous optical fiber, restricting its application. However, this technique was modified to non-invasive irradiation through the radial artery, now called vascular photobiomodulation (VPBM). Many studies have used both, ILIB and VPBM, to treat lung diseases. It is well established that lung diseases affect more than 300 million people worldwide with high morbidity and mortality rates. In this short critical review, we discuss the potential benefits of photobiomodulation to treat lung diseases using these two approaches. The search was performed in the electronic database of MEDLINE (Medical Literature Analysis and Retrieval System Online) via PubMed. The data search was carried out from 1991 to 2017. We selected a total of 10 clinical studies using either ILIB or VPBM, in addition to 2 experimental studies in animals. The respiratory diseases treated in these studies included bronchitis, asthma, pneumonia, and tuberculosis. The results showed overall beneficial effects on lung diseases, characterized by a reduction in the inflammatory cascade and antioxidant effects, improvement of hemodynamic parameters, the efficiency of gas exchange, and reduction of hospitalization periods. In conclusion, all studies showed promising effects of ILIB in both animal and human studies. The studies did not discuss any disadvantages or contraindications. However, further studies are needed in order to understand the dosimetry, and the literature is lacking in randomized, controlled clinical trials. Thus, this review highlights the need for additional studies using this approach.

Sarcopenic Obesity in People with Alcoholic Use Disorder: Relation with Inflammation, Vascular Risk Factors and Serum Vitamin D Levels.

In recent years, the terms sarcopenia, sarcopenic obesity, and osteosarcopenic obesity (OSO) were coined to define a situation in elderly people strongly associated with frailty and increased mortality. Possibly, a complex interplay of several hormones and cytokines are involved in its development. Ongoing research detected that OSO may occur at any age and in several conditions. The prevalence of OSO in alcoholism was poorly analyzed. The aim of this study was to analyze the prevalence of OSO in alcoholism and its relationship with proinflammatory cytokines and/or common complications of alcoholism, such as cirrhosis, cancer, or vascular disease. We included 115 patients with alcoholic use disorder. Body composition analysis was performed by double X-ray absorptiometry. Handgrip strength was recorded using a dynamometer. We assessed liver function according to Child's classification, and determined serum levels of proinflammatory cytokines (TNF-α, IL-6, IL-8), routine laboratory variables, and vitamin D. People with alcoholic use disorder showed a high prevalence of OSO, especially regarding OSO obesity (60%), OSO osteopenia (55.65%), and OSO lean mass (60.17%). OSO handgrip was closely, independently, related to the presence of vascular calcification (χ2 = 17.00; p < 0.001). OSO handgrip was related to several proinflammatory cytokines and vitamin D. Vitamin D deficiency kept a close correlation with OSO handgrip (rho = -0.54, p < 0.001). Therefore, among people with alcohol use disorder, OSO prevalence was high. OSO handgrip is related to serum proinflammatory cytokine levels supporting the possible pathogenetic role of these cytokines on OSO development. Vitamin D deficiency is related to OSO handgrip suggesting its pathogenetic involvement in sarcopenia in patients with alcohol use disorder. The close association between OSO handgrip and vascular calcification is clinically relevant and suggests that OSO handgrip may constitute a prognostic tool in these patients.

Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

Transcutaneous systemic photobiomodulation reduced lung inflammation in experimental model of asthma by altering the mast cell degranulation and interleukin 10 level.

Asthma is a chronic inflammatory disease characterized by recurrent and reversible episodes of wheezing, dyspnea, chest stiffness, and cough. Its treatment includes several drugs, high cost, and considerable side effects. Photobiomodulation (PBM) emerges as an alternative treatment, showing good results, and it can be applied locally or systemically. Here, we aim to evaluate the effect of transcutaneous systemic photobiomodulation (TSPBM) by red diode light. Therefore, adult rats were sensitized and challenged with ovalbumin (OVA) plus alum for induction of asthma and irradiated or not with TSPBM in the caudal vein (wavelength 660 ± 10 nm; total radiant emission 15 J; area 2.8 cm2; energy density 5.35 J/cm2; irradiance 33.3 mW/cm2; exposure time 150 s). Our investigations prioritized the cell migration into the alveolar space and lung, tracheal responsiveness, release and gene expression of cytokines, mast cell degranulation, and anaphylactic antibodies. Our results showed that TSPBM reduced the cell migration and mast cell degranulation without altering the tracheal responsiveness and ovalbumin antibody titers. Indeed, TSPBM increased the levels of interleukin 10 (IL-10) in the BAL fluid without altering the gene expression of cytokines in the lung tissue. Thus, this study showed that transcutaneous systemic irradiation reduced lung inflammation by altering mast cells degranulation and IL-10 level. Considering that this study is a pioneer in the used of light by the systemic route to treat asthma, the data are interesting and instigate future investigations, mainly in relation to the mechanisms involved and in dosimetry.

Local (but not systemic) photobiomodulation treatment reduces mast cell degranulation, eicosanoids, and Th2 cytokines in an experimental model of allergic rhinitis.

Allergic rhinitis (AR) is an inflammatory disorder of the nasal mucosa, and is a worldwide health problem with a significant impact on the quality of life. The main goal of AR treatment is to relieve symptoms. However, standard treatments have considerable side effects or are not effective. Photobiomodulation (PBM) therapy has emerged as an alternative treatment. Here, we evaluated the effects of transcutaneous systemic (tail) or local (skin over nostrils) PBM using a 660-nm light-emitting diode (LED) array. Adult rats were assigned into 4 groups: basal, as non-manipulated animals; Sham, as rats sensitized with 7 intradermal injections of ovalbumin (OVA) plus alum followed by intranasal instillation with OVA (2%) daily for 7 days; and the LPBM and SPBM groups, in which the animals were treated with PBM (local or systemic) immediately after the last instillation of OVA (1%) daily for 3 days. Our results showed that local PBM treatment reduced mast cell degranulation in the nasopharynx and nostrils; levels of leukotriene B4, thromboxane A2, and interleukin 4 (IL-4) in the nasopharynx; and gene expression of IL-4. Moreover, we showed higher levels and gene expression of IL-10 after local PBM treatment. Systemic PBM treatment did not change any of the evaluated parameters. In conclusion, our data showed that local (but not systemic) treatment with PBM could improve parameters related to AR in an animal model, and should be tested clinically.

Beneficial effects of infrared light-emitting diode in corticosteroid-resistant asthma.

Corticosteroid-resistant asthma (CRA) is a severe form of disease and clinically important, since patients do not respond to mainstay corticosteroid therapies. Thus, new therapies are needed. However, a big limiting factor in the understanding of CRA is the existence of different immunological and inflammatory phenotypes, a fact that makes it difficult to reproduce experimentally. Photobiomodulation (PBM) emerges as an alternative therapy based on earlier studies. This study aims to evaluate the effect of PBM using infrared light-emitting diode (ILED) on the development of corticosteroid-resistant asthma. Therefore, groups of rats were sensitized and challenged with ovalbumin plus Freund's adjuvant for the induction of CRA, and treated or not with ILED directly in the respiratory tract on the skin (wavelength 810 nm; power 100 mW; density energy 5 J/cm; total energy 15 J; time 150 s). Our experimental model was capable to induce neutrophilic asthma. Besides that, the corticosteroid treatment did not reverse the lung cell migration as well as the levels of leukotriene B4, and interleukins 17 and 6. The treatment with ILED reduced the lung cell migration; myeloperoxidase activity; mast cell degranulation; and the levels of leukotriene B4, thromboxane B2, prostaglandin E2, tumoral necrosis factor alpha, and interleukins 17 and 6. Still, ILED increased the level of interleukin 10. In conclusion, we showed promisor effects of ILED when irradiated directly in the respiratory tract as adjuvant treatment of corticosteroid-resistant asthma.

Pharmacological doses of melatonin impede cognitive decline in tau-related Alzheimer models, once tauopathy is initiated, by restoring the autophagic flux.

Alterations in autophagy are increasingly being recognized in the pathogenesis of proteinopathies like Alzheimer's disease (AD). This study was conducted to evaluate whether melatonin treatment could provide beneficial effects in an Alzheimer model related to tauopathy by improving the autophagic flux and, thereby, prevent cognitive decline. The injection of AAV-hTauP301L viral vectors and treatment/injection with okadaic acid were used to achieve mouse and human ex vivo, and in vivo tau-related models. Melatonin (10 µmol/L) impeded oxidative stress, tau hyperphosphorylation, and cell death by restoring autophagy flux in the ex vivo models. In the in vivo studies, intracerebroventricular injection of AAV-hTauP301L increased oxidative stress, neuroinflammation, and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment (10 mg/kg), starting once the alterations mentioned above were established (from day 7 to day 28), reduced oxidative stress, neuroinflammation, tau hyperphosphorylation, and caspase-3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how administration of pharmacological doses of melatonin, once tauopathy is initiated, can restore the autophagy flux, reduce proteinopathy, and prevent cognitive decline. We therefore propose exogenous melatonin supplementation or the development of melatonin derivatives to improve autophagy flux for the treatment of proteinopathies like AD.

High-valent iron (Fe(VI), Fe(V), and Fe(IV)) species in water: characterization and oxidative transformation of estrogenic hormones.

This paper presents solid state synthesis and characterization of tetra-oxy iron(iv) and iron(v) species in their salt forms (Na4FeO4-Fe(IV) and K3FeO4-Fe(V)). Stability of the synthesized salts, commonly called ferrates, in water was determined by applying the (57)Fe Mössbauer spectroscopy technique. Within 2 s in water, Fe(IV) converted into Fe(III) while Fe(V) transformed into Fe(VI) and Fe(III) at pH = 8.2. Comparatively, Fe(VI) (bought as K2FeO4) remained stable in aqueous solution during the short time period. The oxidative removal efficiency of the high-valent iron species was then tested against five environmentally important estrogenic hormones (estron (E1), 17-ß-estradiol (E2), estriol (E3), 17-α-ethinylestradiol (EE2), and diethylstibestrol (DES)) in effluent water of a wastewater treatment plant. Three dosages of iron species (1, 10, and 100 mg L(-1)) were applied to the effluent water. An increase in the concentration of dosages enhanced the removal of estrogens. Both Fe(V) and Fe(VI) were effective in degrading estrogens, but Fe(IV) showed limited oxidation capacity to transform estrogens. The oxidized products of the estrogens were analyzed using Raman spectroscopy and high-performance liquid chromatography-mass spectrometry (HPLC-MS) techniques. Results demonstrated the transformation of estrogens into low molecular weight oxygenated compounds such as quinone-like and opened-aromatic ring species. A detailed study on E1 by using excess Fe(VI) showed the mineralization of the parent compound. The results demonstrate great potential of high-valent iron species in the degradation of endocrine disruptor chemicals like estrogens with several superior aspects including fast reactions, complete degradation and/or formation of benign organic species, and environmentally-acceptable iron oxide by-products.

MicroRNA-31 Emerges as a Predictive Biomarker of Pathological Response and Outcome in Locally Advanced Rectal Cancer.

Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision has emerged as the standard treatment for locally advanced rectal cancer (LARC) patients. However, many cases do not respond to neoadjuvant CRT, suffering unnecessary toxicities and surgery delays. Thus, identification of predictive biomarkers for neoadjuvant CRT is a current clinical need. In the present study, microRNA-31 expression was measured in formalin-fixed paraffin-embedded (FFPE) biopsies from 78 patients diagnosed with LARC who were treated with neoadjuvant CRT. Then, the obtained results were correlated with clinical and pathological characteristics and outcome. High microRNA-31 (miR-31) levels were found overexpressed in 34.2% of cases. Its overexpression significantly predicted poor pathological response (p = 0.018) and worse overall survival (OS) (p = 0.008). The odds ratio for no pathological response among patients with miR-31 overexpression was 0.18 (Confidence Interval = 0.06 to 0.57; p = 0.003). Multivariate analysis corroborated the clinical impact of miR-31 in determining pathological response to neoadjuvant CRT as well as OS. Altogether, miR-31 quantification emerges as a novel valuable clinical tool to predict both pathological response and outcome in LARC patients.

PP2A inhibition as a novel therapeutic target in castration-resistant prostate cancer.

Protein phosphatase 2A (PP2A) is a well-known tumor suppressor frequently inhibited in human cancer. Alterations affecting PP2A subunits together with the deregulation of endogenous PP2A inhibitors such as CIP2A and SET have been described as contributing mechanisms to inactivate PP2A in prostate cancer. Moreover, recent findings highlight that functional inactivation of PP2A could represent a key event in the acquisition of castration-resistant phenotype and a novel molecular target with high impact at both clinical and therapeutic levels in prostate cancer.

Activation of the Tumor Suppressor PP2A Emerges as a Potential Therapeutic Strategy for Treating Prostate Cancer.

Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients.

Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma.

Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

Omega-3 and Omega-6 Polyunsaturated Fatty Acid Intakes, Determinants and Dietary Sources in the Spanish Population: Findings from the ANIBES Study.

The multiple roles of polyunsaturated fatty acids (PUFA) in growth and general health are well documented. However, available intake data for the Spanish population are limited and lack gender and age considerations. Therefore, our goal was to assess dietary intake adequacy of omega-3 and omega-6 PUFA, their determinants and their major food sources among the Spanish population. Due to their influence on various beneficial functions attributed to omega-3 PUFA, combined intake adequacy with folic acid (FA), vitamin B12 and choline was also assessed. Intake data were obtained from the ANIBES cross-sectional study on a representative sample of the Spanish population (9-75 years; n = 2009), where dietary intake was analysed with a three-day dietary record. Median intake of total omega-3 PUFA stood at 0.81 g/day (0.56-1.19 g/day), with α-linolenic acid (ALA) at 0.61 g/day (0.45-0.85 g/day), eicosapentaenoic acid (EPA) at 0.03 g/day (0.01-0.12 g/day) and docosahexaenoic acid (DHA) at 0.06 g/day (0.0-0.20 g/day). Accordingly, 65% of the Spanish population showed insufficient intakes for total omega-3 PUFA; 87% for ALA, and 83% for combined EPA and DHA. Inadequate intakes were significantly higher in children, adolescents, and younger women of childbearing age (18-30 years). In contrast, inadequacy due to excessive intakes was almost negligible. Regarding omega-6 PUFA, total intake was 10.1 g/day (7.0-14.0 g/day), 10.0 g/day (6.9-13.9 g/day) for linoleic acid (LA) and 0.08 g/day (0.05-0.13 g/day) for arachidonic acid (AA). Non-compliance due to either insufficient or excessive intakes of LA stood at around 5% of the sample, with the elderly showing significantly higher degrees of inadequacy due to insufficient intakes (10%; p ≤ 0.05). Median omega-6 to omega-3 ratio was 12:1, and significantly higher in men compared to women (p ≤ 0.05); in children, adolescents and adults compared to the elderly (p ≤ 0.05); and in younger women of childbearing age compared to the older group (31-45 years) (p ≤ 0.001). Oils and fats and meat and meat products were the main dietary sources for the essential fatty acids LA and ALA, respectively. Meat and meat products were as well the main providers of AA, while fish and shellfish were almost exclusively the only sources of EPA and DHA. However, main food sources identified showed important differences across age groups. Finally, the total combined degree of inadequacy observed for omega-3 PUFA, FA, vitamin B12 and choline reached 21.3% of the ANIBES population. The observed degree of inadequacy of omega-3 PUFA intakes among the Spanish population makes it urgent to increase its consumption and to consider the need for supplementation. This should also be the main strategy for the optimization of the omega-6/omega-3 ratio, as the adequacy observed for omega-6 intakes is relatively acceptable. Additional improvement of the dietary intake of FA, vitamin B12 and choline could contribute to the beneficial effects of omega-3 PUFA.

Dietary intake, nutritional adequacy and food sources of vitamins involved in the methionine-methylation cycle from Spanish children aged one to <10 years: results from the EsNuPI study.

Background: Methionine-methylation cycle and the derived critical functions during infancy are key regulated by folates, vitamins B12, and B6. At present in Spain, there is an absence of studies that assess the intakes and dietary sources of total folates and B12 by children consuming all types of milks and those regularly consuming adapted milk formulas. Thus, our aim was to evaluate folates intakes alongside with vitamins B6 and B12 while describing their major dietary contributors in Spanish children aged one to <10 years. Methods: A total of 1,448 children aged between 1 and 10 years (49.7% girls and 50.3% boys) from the EsNuPI, a prospective cross-sectional study, were allocated into two cohorts: one Spanish Reference Cohort (SRS) of the general population (n = 707), and another including children consuming adapted milks called Adapted Milk Consumers Cohort (AMS) (n = 741) completed two 24 h dietary recalls used to estimate their nutrient intakes and to compare them to the European Food Safety Authority (EFSA) Population Reference Intakes. Results: The median intake of vitamin B6 was 1.35 (1.06-1.70) mg/day in the SRS and 1.45 (1.17-1.79) mg/day in the AMS, being significantly higher in the AMS for all age-groups. Prevalence of adequacy for vitamin B6 in the SRS and AMS was 97.7 and 98.7%, respectively. Total folates intakes in the AMS were significantly higher (p ≤ 0.001) in all age groups than in the SRS, independently of age. In addition, the prevalence of adequacy for folates intakes in all groups was more than 60%. Vitamin B12 intake increased with age independently of the type of milk consumed. The prevalence of adequacy for vitamin B12 was highly compliant by all population groups. The major contributors to vitamin B6 were milk and dairy products being significantly higher in AMS than SRS (p ≤ 0.001). The highest contributors to folates intakes were milk and dairy products, cereals, vegetables, and fruits in both groups whereas for vitamin B12 in the SRS sample were milk and dairy products followed by meat and meats products and for adapted milks, were milk and dairy products, followed by eggs, then meat and meats products. Conclusion: A satisfactory prevalence of adequacy for vitamins B6, and B12 amongst the Spanish children population was observed, which was not the case for folates, regardless of the dietary group evaluated. Nevertheless, a possible strategy to increase folate intake among the youngest children is to increase the consumption of milk and dairy products within a healthier dietary pattern, as these may contribute significantly to the vitamin needs of the infant population.

Dietary Intake of Individual (Intrinsic and Added) Sugars and Food Sources from Spanish Children Aged One to <10 Years­Results from the EsNuPI Study

Currently, in Spain there are no studies assessing the intakes and sources of intrinsic and added sugars by both children consuming standard milks and children regularly consuming adapted milk formulas. Our goal was to evaluate current sugar intake levels (intrinsic and added) and their major dietary sources within the EsNuPI study participants by applying two 24-h dietary recalls that were completed by 1448 children (1 to <10 years) divided into two subsamples: One "Spanish Reference Sample" (SRS) of the general population (n = 707) and another sample which included children consuming adapted milks including follow-on milk, toddler's or growing up milk and fortified and enriched milks, here called "Adapted Milk Consumers Sample" (AMS) (n = 741). Estimates of intrinsic and added sugar intakes from the Spanish EsNuPI population as well as the adherence to recommendations varied notably according to age segment, but no major differences between subsamples were found. Younger children (1 to <3 years) showed the highest added sugar contribution to total energy intake (TEI) (SRS: 12.5% for boys and 11.7% for girls; AMS: 12.2% for boys and 11.3% for girls) and the lowest adherence to recommendations set at <10% TEI (SRS: 27.4% for boys and 37.2% for girls; AMS: 31.3% for boys and 34.7% for girls). Adherence increased with age but remains inadequate, with approximately one in two children from the older age segment (6 to <10 years) exceeding the recommendations. Main food sources of intrinsic sugars for both subsamples were milk and dairy products, fruits, vegetables and cereals, while for added sugars, these were milk and dairy products (mainly yogurts), sugars and sweets (mainly sugary cocoa and nougat), bakery products (mainly cookies) and cereals (mainly bread and wheat flour). However, for the AMS, the groups milk and dairy products and cereals showed a significantly lower contribution to intrinsic sugar intake but a significantly higher contribution to that of added sugars. These results demonstrate that sugar intake and the adherence to recommendations in the studied population varied notably according to age but not to the type of milk consumed. In addition, our results highlight the need to monitor the consumption of added sugars by the infant population, as well as the need to make efforts to facilitate this task, such as harmonizing the recommendations regarding free/added sugars and the inclusion of information on their content on the nutritional labeling of products in order to incorporate them into food composition databases.

Implication of type 4 NADPH oxidase (NOX4) in tauopathy.

Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.