Understanding consumer food choices & promotion of healthy and sustainable Mediterranean diet and lifestyle in children and adolescents through behavioural change actions: the DELICIOUS project.

Over the last decades, the abandonment of traditional dietary patterns, such as the Mediterranean diet, represents an important threat for human health and environmental safeguard. The DELICIOUS project aims to promote healthy lifestyles among children and adolescents by implementing activities and tools to increase the adherence to the Mediterranean Diet with an attention to the environmental impacts of the diet. This study protocol describes the DELICIOUS project as a single-arm, uncontrolled behavioural intervention providing formal and non-formal education activities, development of new snacks and recipe reformulation, web/mobile app development, and physical activities to school children and adolescents in five European countries. The project aims to increase awareness of the nutritional benefits and the sustainability aspects of the Mediterranean Diet and to promote consumers' empowerment through an online platform for sustainable and healthy meal planning in the school canteen.

Novel Foscan®-derived ring-fused chlorins for photodynamic therapy of cancer.

Photodynamic therapy (PDT) is an established anticancer treatment that combines the use of a photosensitiser (PS) and a light source of a specific wavelength for the generation of reactive oxygen species (ROS) that are toxic to the tumour cells. Foscan® (mTHPC) is a clinically-approved chlorin used for the PDT treatment of advanced head and neck, prostate and pancreatic cancers but is characterized by being photochemically unstable and associated with prolonged skin photosensitivity. Herein, we report the synthesis of new 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, having the meso-tetra(3-hydroxyphenyl)macrocycle core of mTHPC, by exploring the [8π + 2π] cycloaddition of a meso-tetra(3-hydroxyphenyl)porphyrin derivative with diazafulvenium methides. These chlorins have photochemical properties similar to Foscan® but are much more photostable. Among the novel compounds, two chlorins with a hydroxymethyl group and its azide derivative present in the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused system, are promising photodynamic agents with activity in the 100 nM range against triple-negative breast cancer cells and, in the case of azidomethyl chlorin, a safer phototherapeutic index compared to Foscan®.

Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid ß protein precursor (APP) and neurotoxic amyloid ß (Aß) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aß peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.

Evaluation of mutagenesis, necrosis and apoptosis induced by omeprazole in stomach cells of patients with gastritis.

BACKGROUND: Gastritis is a superficial and prevalent inflammatory lesion that is considered a public health concern once can cause gastric ulcers and gastric cancer, especially when associated with Helicobacter pylori infection. Proton pump inhibitors, such as omeprazole, are the most widely used drugs to treat this illness. The aim of the study was evaluate cytogenetic effects of omeprazole in stomach epithelial cells of patients with gastritis in presence and absence of H. pylori, through cytogenetic biomarkers and catalse and superoxide dismutase analysis. METHODS: The study included 152 patients from the Gastroenterology Outpatient Clinic of Hospital Getúlio Vargas, Teresina-Brazil, that reported continuous and prolonged omeprazole use in doses of 20, 30 and 40 mg/kg. The participants were divided into groups: (1) patients without gastritis (n = 32); (2) patients without gastritis but with OME use (n = 24); (3) patients with gastritis (n = 26); (4) patients with gastritis undergoing OME therapy (n = 26); (5) patients with gastritis and H. pylori (n = 22) and (6) patients with gastritis and H. pylori on OME therapy (n = 22). RESULTS: OME induced cytogenetic imbalance in the stomach epithelium through the formation of micronuclei (group 6 > 1, 2, 3, 4, 5; group 5 > 1, 2, 3; group 4 > 1, 2, 3); bridges (groups 4 and 6 > 1, 2, 3, 5 and group 2 > 3, 5); buds (groups 2,4,6 > , 1, 3, 5); binucleated cells (group 6 > 1, 2, 3, 4, 5; group 4 > 1, 2, 3); (groups 2 and 3 > 1); picnoses (group 6 > 1, 2, 3, 4, 5), groups 2 and 5 > 1, 3; group 4 > 1, 2, 3, 5); cariorrexis (groups 6 and 4 > 1, 2, 3, 5; groups 2, 3, 5 > 1) and karyolysis (groups 2, 4, and 6 > 1, 3, 5; groups 3 and 5 > 1). The OME cytogenetic instability was associated with H. pylori infection, indicating clastogenic/aneugenic effects, chromosomes alterations, gene expression changes, cytotoxicity and apoptosis. CONCLUSIONS: The cytogenetic changescan be attributed to several mechanisms that are still unclear, including oxidative damage, as observed by increased catalase and superoxide dismutase expresion. Positive correlations between antioxidant enzymes were found with micronuclei formation, and were negative for picnoses. Thus, the continuous and prolonged omeprazole use induces genetic instability, which can be monitored through cytogenetic analyzes, as precursor for gastric cancer.

Cytogenotoxic evaluation of the acetonitrile extract, citrinin and dicitrinin-A from Penicillium citrinum.

Endophytic fungi are promising sources of bioactive substances; however, their secondary metabolites are toxic to plants, animals, and humans. This study aimed toevaluate the toxic, cytotoxic, mutagenic and oxidant/antioxidant activities of acetonitrile extract (AEPc), citrinin (CIT) and dicitrinin-A (DIC-A) of Penicillium citrinum. For this, the test substances at 0.5; 1.0; 1.5 and 2 µg/mLwere exposed for 24 and 48 h in Artemia salina, and 48 h in Allium cepa test systems. The oxidant/antioxidant test was evaluated in pre-, co- and post-treatment with the stressor hydrogen peroxide (H2O2) in Saccharomyces cerevisiae. The results suggest that the AEPc, CIT and DIC-A at 0.5; 1.0; 1.5 and 2 µg/mL showed toxicity in A. saline, with LC50 (24 h) of 2.03 µg/mL, 1.71 µg/mL and 2.29 µg/mL, and LC50 (48 h) of 0.51 µg/mL, 0.54 µg/mL and 0.54 µg/mL, respectively.In A. cepa, the test substances also exerted cytotoxic and mutagenic effects. The AEPc, CIT and DIC-A at lower concentrations modulated the damage induced by H2O2 in the proficient and mutant strains of S. cerevisiae for cytoplasmic and mitochondrial superoxide dismutase. Moreover, the AEPc at 2 µg/mL and CIT at the two highest concentrations did not affect the H2O2-induced DNA damage in the test strains. In conclusion, AEPc, CIT and DIC-A of P. citrinum may exert their toxic, cytotoxic and mutagenic effects in the test systems possibly through oxidative stress induction pathway.

Special Issue "Molecular and Cellular Mechanisms of Action of Markers of Tissue Degeneration".

Tissue degeneration is an event shared by many, if not all, age-related pathologies [...].

Design and Experimental Evaluation of a Peptide Antagonist against Amyloid ß(1-42) Interactions with Calmodulin and Calbindin-D28k.

Amyloid ß1-42 (Aß(1-42)) oligomers have been linked to the pathogenesis of Alzheimer's disease (AD). Intracellular calcium (Ca2+) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aß neurotoxicity in AD. The Ca2+ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aß(1-42) oligomers and extensively binds internalized Aß(1-42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aß (1-42), using a combined strategy based on the experimental results obtained for Aß(1-42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aß(1-42) HiLyteTM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aß(1-42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aß(1-42):CaM and of Aß(1-42):calbindin-D28k complexes.

Conformational Dynamics of the Soluble and Membrane-Bound Forms of Interleukin-1 Receptor Type-1: Insights into Linker Flexibility and Domain Orientation.

Interleukin-1 receptor type 1 (IL-1R1) is a key player in inflammation and immune responses. This receptor regulates IL-1 activity in two forms: as a membrane-bound form and as a soluble ectodomain. The details and differences between the conformational dynamics of the membrane-bound and the soluble IL-1R1 ectodomains (ECDs) remain largely elusive. Here, we study and compare the structural dynamics of the soluble and membrane-bound IL-1R1-ECDs using molecular dynamics (MD) simulations, focusing on the flexible interdomain linker of the ECD, as well as the spatial rearrangements between the Ig-like domains of the ECD. To explore the membrane-bound conformations, a full-length IL-1R1 structural model was developed and subjected to classical equilibrium MD. Comparative analysis of multiple MD trajectories of the soluble and the membrane-bound IL-1R1-ECDs reveals that (i) as somewhat expected, the extent of the visited "open-to-closed" transitional states differs significantly between the soluble and membrane-bound forms; (ii) the soluble form presents open-closed transitions, sampling a wider rotational motion between the Ig-like domains of the ECD, visiting closed and "twisted" conformations in higher extent, whereas the membrane-bound form is characterized by more conformationally restricted states; (iii) interestingly, the backbone dihedral angles of residues Glu202, Glu203 and Asn204, located in the flexible linker, display the highest variations during the transition between discrete conformational states detected in IL-1R1, thus appearing to work as the "central wheel of a clock's movement". The simulations and analyses presented in this contribution offer a deeper insight into the structure and dynamics of IL-1R1, which may be explored in a drug discovery setting.

Internalized Amyloid-ß (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells.

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-ß peptides (Aß) in AD. In recent works, we have proposed that Aß-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 µM Aß(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aß(1-42), respectively. Internalized Aß(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aß. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aß(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aß(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aß(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 µM Aß(1-42) oligomers. We conclude that Aß(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.

Spent coffee grounds by-products and their influence on soil C-N dynamics.

SCG are a bio-waste generated in great amount worldwide which are attractive as soil amendment for their high content of organic matter and nutritive elements. Nevertheless, several studies have shown that soil application of untreated SCG has detrimental agronomic and environmental effects due to their high degradability and content of noxious compounds (phenols, caffeine, and tannins). However, SCG can be valorised, in the frame of circular economy, by extraction of energy and valuable products (carbohydrates, proteins, bio-oil, bio-diesel) and generation of solid by products (biochar, hydrochar, compost) that can be utilized as soil fertilizers and amendments. Therefore, the aim of this work was the characterization of different solid SCG by-products (as second-generation products) and their assessment as effective organic amendments. The novelty of this study is that for the first time 8 different by-products derived from the same SCG were characterized and comparatively evaluated for their impact on the C and N cycles of soil. SCG was collected and treated to generate 8 different SCG by-products (biochars produced at 270 and 400 °C, hydrochars produced at 160 and 200 °C, vermicompost, defatted SCG and biochars produced from defatted SCG at 270 and 400 °C). SCG and derived by-products were characterized for SEM micromorphology, pH and EC values, and C, N, H, O, volatile matter, fixed C, LOI, carbonates, water soluble C and N, NO3- and NH4+ content. SCG and SCG by-products assessment as organic amendments was performed with an incubation experiment. The residues were added (2.5%) to a moist Mediterranean agricultural soil and the amended soil samples were placed in mesocosms and incubated at 20 °C for 30 days. During incubation, CO2 and N2O emissions were measured every 6 h by means of a gas chromatography automated system for GHG sampling and measurement. The percentage of added C remaining (CR) in the soil was calculated by fitting the cumulative respiration of amended soil to a two-pool model. After 2, 7 and 30 days of incubation, the control and amended soils were sampled and analyzed for their content of extractable organic C, N, NO3- and NH4+ and microbial biomass C and N. Results showed that SCG by-products presented a great variability in their properties. SCG and hydrochars presented higher contents in volatile matter and water soluble C and N, and low content of fixed C, while biochars showed an opposite behaviour. SEM images confirmed the different characteristics of the SCG by-products: the biochar presented a porous structure, honeycomb-like form, due to the loss of the more soluble compounds, while the SCG and hydrochars' pores were filled with amorphous carbonaceous materials. Consequently, soil addition of SCG by-products showed a distinct impact on C and N cycle and microbial biomass content. Addition of SCG and hydrochars generated the highest cumulative CO2-C emissions (2103-2300 µg g-1), the lower amount of CR (86.8-88.6%), increased the soil extractable organic C and microbial biomass C and N and caused N immobilization. On the other hand, the addition of biochars generated lower CO2-C emissions (542-1060 µg g-1), higher amounts of CR (96. 3-99.9%) and lower amounts of extractable compounds and microbial biomass C and N, generating also N immobilization, but to a lesser extent. The addition of vermicompost generated 723 µg g-1 of CO2-C and 98% of CR remaining. However, this by-product did not generate N immobilization being able to act as N fertilizer. None of the residues generated N2O emissions. The different properties of the SCG by-products and their impact on C and N cycle indicated that they can be effectively applied to soil to exert different agronomical and environmental functions.

Seeds as Potential Sources of Phenolic Compounds and Minerals for the Indian Population.

Seeds are major sources of nutrients and bioactive compounds for human beings. In this work, the chemical composition and physicochemical properties of 155 Indian seeds (belonging to 49 families) are reported. Moisture and ash were measured with reference protocols from AOAC; total polyphenols and flavonoids were measured with spectrophotometric methods after extraction with organic solvents, and mineral elements were determined by X-ray fluorescence spectrophotometry. Total phenolic compounds, flavonoids and mineral contents (Al, Ba, Ca, Cl, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, P, Rb, S, Sr, Ti, V and Zn) were found to vary in the ranges 182−5000, 110−4465 and 687−7904 mg/100 g (DW), respectively. Noticeably, polyphenol contents higher than 2750 mg/100 g were observed in 18 seeds. In addition, mineral contents >5000 mg/100 g were detected in the seeds from Cuminum cyminum, Foeniculum vulgare, Commiphora wightii, Parkia javanica, Putranjiva roxburghii, Santalum album and Strychnos potatorum. Botanical and taxonomical variations in the proximate characteristics of the examined seeds are also discussed.

The endoplasmic reticulum Ca2+ -ATPase SERCA2b is upregulated in activated microglia and its inhibition causes opposite effects on migration and phagocytosis.

Activation of microglia is an early immune response to damage in the brain. Although a key role for Ca2+ as trigger of microglial activation has been considered, little is known about the molecular scenario for regulating Ca2+ homeostasis in these cells. Taking into account the importance of the endoplasmic reticulum as a cellular Ca2+ store, the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA2b) is an interesting target to modulate intracellular Ca2+ dynamics. We found upregulation of SERCA2b in activated microglia of human brain with Alzheimer's disease and we further studied the participation of SERCA2b in microglial functions by using the BV2 murine microglial cell line and primary microglia isolated from mouse brain. To trigger microglia activation, we used the bacterial lipopolysaccharide (LPS), which is known to induce an increase of cytosolic Ca2+ . Our results showed an upregulated expression of SERCA2b in LPS-induced activated microglia likely associated to an attempt to restore the increased cytosolic Ca2+ concentration. We analyzed SERCA2b contribution in microglial migration by using the specific SERCA inhibitor thapsigargin in scratch assays. Microglial migration was strongly stimulated with thapsigargin, even more than with LPS-induction, but delayed in time. However, phagocytic capacity of microglia was blocked in the presence of the SERCA inhibitor, indicating the importance of a tight control of cytosolic Ca2+ in these processes. All together, these results provide for the first time compelling evidence for SERCA2b as a major player regulating microglial functions, affecting migration and phagocytosis in an opposite manner.

Citrinin against breast cancer: A cytogenotoxicological study.

Breast cancer is one of the most lethal types of cancer and a leading cause of mortality among Women worldwide. Citrinin (CIT), a polyketide extracted from the fungus Penicillium citrinum, exhibits a wide range of biological activities such as antibacterial, antifungal, and cytotoxic effects. The aim of the current study was to evaluate the antitumoral effects of CIT against 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinoma in Swiss mice For this, CIT, DMBA and the standard cyclophosphamide (CPA) induced behavioral changes in experimental animals, and these changes were screened by using the rota rod and open field tests. Additionally, hematological, biochemical, immuno-histochemical, and histopathological analyses were carried out. Results suggest that CIT did not alter behavioral, hematological, and biochemical parameters in mice. DMBA induced invasive mammary carcinoma and showed genotoxic effects in the breasts, bone marrow, lymphocytes, and hepatic cells. It also caused mutagenic effects in the formation of micronuclei, bridges, shoots, and binucleate cells in bone marrow and liver. CIT and CPA genotoxic effects were observed after 3 weeks of therapy, where CIT exhibited a repair capacity and induced significant apoptotic damage in mouse lymphocytes. In conclusion, CIT showed antitumoral effects in Swiss mice, possibly through induction of apoptosis.

The Antioxidant Transcription Factor Nrf2 in Cardiac Ischemia-Reperfusion Injury.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that controls cellular defense responses against toxic and oxidative stress by modulating the expression of genes involved in antioxidant response and drug detoxification. In addition to maintaining redox homeostasis, Nrf2 is also involved in various cellular processes including metabolism and inflammation. Nrf2 activity is tightly regulated at the transcriptional, post-transcriptional and post-translational levels, which allows cells to quickly respond to pathological stress. In the present review, we describe the molecular mechanisms underlying the transcriptional regulation of Nrf2. We also focus on the impact of Nrf2 in cardiac ischemia-reperfusion injury, a condition that stimulates the overproduction of reactive oxygen species. Finally, we analyze the protective effect of several natural and synthetic compounds that induce Nrf2 activation and protect against ischemia-reperfusion injury in the heart and other organs, and their potential clinical application.

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer's Disease on the Pump Activity.

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer's disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-ß peptide (Aß) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aß and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aß and tau on the pump, and counteracts the neurotoxicity of Aß and tau by interacting with them.

The Relevance of Amyloid ß-Calmodulin Complexation in Neurons and Brain Degeneration in Alzheimer's Disease.

Intraneuronal amyloid ß (Aß) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer's disease (AD)-affected brains, intraneuronal Aß oligomers can derive from Aß peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aß neurotoxicity in AD. However, the identification of primary Aß-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aß peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aß concentrations in the low nanomolar range. In turn, the concentration of Aß-CaM complexes within neurons will increase as a function of time after the induction of Aß production, and free Aß will rise sharply when accumulated Aß exceeds all available CaM. Thus, Aß-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aß; a point that has been overlooked until now. In this review, we address the implications of Aß-CaM complexation in the formation of neurotoxic Aß oligomers, in the alteration of intracellular calcium homeostasis induced by Aß, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aß.

Binding of Amyloid ß(1-42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis.

Lipid rafts are a primary target in studies of amyloid ß (Aß) cytotoxicity in neurons. Exogenous Aß peptides bind to lipid rafts, which in turn play a key role in Aß uptake, leading to the formation of neurotoxic intracellular Aß aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer's disease (AD). In a previous work, we showed that Aß(1-42), the prevalent Aß peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aß(1-42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aß(1-42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aß(1-42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aß(1-42) in lipid rafts in CGN stained with up to 100 picomoles of Aß(1-42)-HiLyteTM-Fluor555 monomers. Intracellular Aß(1-42) concentration in this range was achieved by 2 h incubation of CGN with 2 µM Aß(1-42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aß(1-42) dimers, whose activity is inhibited by CaM:Aß(1-42) complexes bound to lipid rafts.

Junctional sarcoplasmic reticulum motility in adult mouse ventricular myocytes.

Excitation-contraction (EC) coupling is the coordinated process by which an action potential triggers cardiac myocyte contraction. EC coupling is initiated in dyads where the junctional sarcoplasmic reticulum (jSR) is in tight proximity to the sarcolemma of cardiac myocytes. Existing models of EC coupling critically depend on dyad stability to ensure the fidelity and strength of EC coupling, where even small variations in ryanodine receptor channel and voltage-gated calcium channel-α 1.2 subunit separation dramatically alter EC coupling. However, dyadic motility has never been studied. Here, we developed a novel strategy to track specific jSR units in dissociated adult ventricular myocytes using photoactivatable fluorescent proteins. We found that the jSR is not static. Instead, we observed dynamic formation and dissolution of multiple dyadic junctions regulated by the microtubule-associated molecular motors kinesin-1 and dynein. Our data support a model where reproducibility of EC coupling results from the activation of a temporally averaged number of SR Ca2+ release units forming and dissolving SR-sarcolemmal junctions. These findings challenge the long-held view that the jSR is an immobile structure and provide insights into the mechanisms underlying its motility.

BIN1 Induces the Formation of T-Tubules and Adult-Like Ca2+ Release Units in Developing Cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates CaV 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca2+ ]i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated CaV 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of CaV 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased CaV 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more CaV 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca2+ ]i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and CaV 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for CaV 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64.

Palmatine antioxidant and anti-acetylcholinesterase activities: A pre-clinical assessment.

There is evidence that palmatine (PA), an alkaloid isolated from the Guatteria friesiana plant, has some important biological activities, including anti-inflammatory and antidepressant effects. In this study, the antioxidant and anti-acetylcholinesterase (AChE) effects of PA were assessed. The antioxidant capacity was evaluated in vitro and in vivo through 7 distinct assays, and the anti-AChE activity was determined in vitro. The standards, trolox and ascorbic acid were used for the in vitro antioxidant test, while hydrogen peroxide was selected as a stressor for the Saccharomyces cerevisiae test. Additionally, PA was also combined with trolox and ascorbic acid to determine the likelihood of synergistic effects occurrence to what concerns to antioxidant potential. PA exhibited a potent and concentration-dependent antioxidant potential, although a stronger antioxidant activity was stated using the PA + trolox combination. PA was also found to inhibit AChE activity when compared to the negative control. Thus, PA may be viewed as a promissory phytotherapeutic agent to manage oxidative stress-mediated neurological diseases, especially the Alzheimer's and Parkinson's diseases.