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Live cell imaging is a robust method to visualize dynamic cellular structures, especially organelles with network-like structures such as mitochondria. In this regard, mitochondrial dynamics, namely mitochondrial fission and fusion, are highly dynamic processes that regulate mitochondrial size and morphology depending on a plethora of cellular cues. Likewise, lysosome size and distribution may hint at their function and state.Here, we describe how to perform live cell confocal imaging using commercially available organelle dyes (MitoTracker, LysoTracker), followed by either 2D or 3D analyses of mitochondrial morphology/network connectivity and lysosomal morphology using the freely available Mitochondria Analyzer plugin for ImageJ/Fiji., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Takotsubo syndrome (TTS) is currently described as an acute and usually reversible form of systolic dysfunction of the left ventricle, which more frequently affects postmenopausal women after a stressful emotional event. Although TTS is a rare condition in premenopausal women, in recent years, the number of reported cases has increased. This manuscript reports the first case of a TTS several months after delivery in a 22-year-old woman during lactation. It may also emphasize the role of estrogens in the disease pathogenesis., (Copyright © 2022 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca 2+ ) oscillations on an extracellular Ca 2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS., (© 2022. The Author(s).)

The gut microbiota influence neurodevelopment, modulate behavior, and contribute to neurodegenerative disorders. Several studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinson disease (PD) fecal samples. Therefore, we investigated whether A.muciniphila -conditioned medium (CM) could initiate α-synuclein (αSyn) misfolding in enteroendocrine cells (EEC) - a component of the gut epithelium featuring neuron-like properties. We found that A . muciniphila CM composition is influenced by the ability of the strain to degrade mucin. Our in vitro experiments showed that the protein-enriched fraction of mucin-free CM induces RyR-mediated Ca 2+ release and increased mitochondrial Ca 2+ uptake leading to ROS generation and αSyn aggregation. Oral administration of A. muciniphila cultivated in the absence of mucin to mice led to αSyn aggregation in cholecystokinin (CCK)-positive EECs but no motor deficits were observed. Noteworthy, buffering mitochondrial Ca 2+ reverted the damaging effects observed. These molecular insights offer evidence that bacterial proteins can induce αSyn aggregation in EECs., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation., Competing Interests: Nothing to disclose., (© 2021 The Author(s).)

Parkinson's disease (PD) is mainly driven by dopaminergic neuronal degeneration in the substantia nigra pars compacta accompanied by chronic neuroinflammation. Despite being mainly sporadic, approximately 10% of all cases are defined as heritable forms of PD, with mutations in the leucine-rich repeat kinase ( LRRK2 ) gene being the most frequent known cause of familial PD. MicroRNAs (miRNAs or miRs), including miR-335, are frequently deregulated in neurodegenerative diseases, such as PD. Here, we aimed to dissect the protective role of miR-335 during inflammation and/or neurodegenerative events in experimental models of PD. Our results showed that miR-335 is significantly downregulated in different PD-mimicking conditions, including BV2 microglia cells stimulated with lipopolysaccharide (LPS) and/or overexpressing wild-type LRRK2. Importantly, these results were confirmed in serum of mice injected with 1-methyl-1-4-phenyl-1,2,3,6-tetrahydripyridine hydrochloride (MPTP), and further validated in patients with idiopathic PD (iPD) and those harboring mutations in LRRK2 (LRRK2-PD), thus corroborating potential clinical relevance. Mechanistically, miR-335 directly targeted LRRK2 mRNA. In the BV2 and N9 microglia cell lines, miR-335 strongly counteracted LPS-induced proinflammatory gene expression, and downregulated receptor interacting protein 1 (RIP1) and RIP3, two important players of necroptotic and inflammatory signaling pathways. Further, miR-335 inhibited LPS-mediated ERK1/2 activation. LRRK2-Wt-induced proinflammatory gene expression was also significantly reduced by miR-335 overexpression. Finally, in SH-SY5Y neuroblastoma cells, miR-335 decreased the expression of pro-inflammatory genes triggered by α-synuclein. In conclusion, we revealed novel roles for miR-335 in both microglia and neuronal cells that strongly halt the effects of classical inflammatory stimuli or LRRK2-Wt overexpression, thus attenuating chronic neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oliveira, Dionísio, Gaspar, Correia Guedes, Coelho, Rosa, Ferreira, Amaral and Rodrigues.)

Parkinson's disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit-Oxa12-that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.

Although Aspergillus flavus and Aspergillus parasiticus are the main microorganisms of concern in peanuts, due to aflatoxin contamination, several Salmonella outbreaks from this product have been reported over the last ten decades. Thus, it is important to understand the relationship between microorganisms to predict, manage and estimate the diversity in the peanut supply chain. The purpose of this study was to evaluate aflatoxin production during the co-cultivation of Aspergillus section Flavi and Salmonella both isolated from peanuts. Three strains of A. section Flavi: A. flavus producing aflatoxin B, A. flavus non-producing aflatoxin and A. parasiticus producing aflatoxin B and G were co-cultivated with seven serotypes of Salmonella of which six were isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) and one was S. Typhimurium ATCC 14028. First of all, each Salmonella strain was inoculated by pour plate (ca. 5 log cfu/mL) in PDA (potato dextrose agar). Then, each pre-cultured fungus was inoculated in the center of the petri dish. The plates were incubated at 30 °C and the fungal colony diameter was measured once a day for 7 days. As a control each Aspergillus strain was cultivated in the absence of Salmonella culture. All three strains of Aspergillus with absence of Salmonella (control) reached the maximum colony diameter and their growth rate was influenced when co-cultivated (p < 0.05) with all Salmonella serotypes tested. The maximum inhibition in the colony diameter was 20% for A. flavus aflatoxin B producer and A. parasiticus, and 18% for A. flavus non- aflatoxin producer when cultivated with Salmonella. However, no significant difference (p < 0.05) in reduction of colony diameter was observed among the Salmonella serotypes. Aflatoxin production was determined previously, by using the agar plug technique on thin layer chromatography (TLC). The production of aflatoxin G by A. parasiticus in co-cultivation with Salmonella was not observed. On the other hand, A. flavus preserved their characteristics of aflatoxin B production. The quantification of aflatoxin reduction by Salmonella interaction was evaluated using HPLC method. There was a maximum reduction of aflatoxin production of 88.7% and 72.9% in A. flavus and A. parasiticus, respectively, when cultivated with Salmonella. These results indicate that some serotypes of Salmonella may interfere with aflatoxin production and fungal growth of A. flavus and A. parasiticus in the peanut supply chain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, being largely characterized by motor features. MicroRNAs (miRNAs) are small non-coding RNAs, whose deregulation has been associated with neurodegeneration in PD. In this study, miRNAs targeting cell death and/or inflammation pathways were selected and their expression compared in the serum of PD patients and healthy controls. We used two independent cohorts (discovery and validation) of 20 idiopathic PD patients (iPD) and 20 healthy controls each. We also analyzed an additional group of 45 patients with a mutation in the leucine-rich repeat kinase 2 ( LRRK2 ) gene (LRRK2-PD). miRNA expression was determined using Taqman qRT-PCR and their performance to discriminate between groups was assessed by receiver operating characteristic (ROC) curve analysis. We found miR-146a, miR-335-3p, and miR-335-5p downregulated in iPD and LRRK2-PD patients versus controls in both cohorts. In addition, miR-155 was upregulated in LRRK2-PD compared to iPD patients showing an appropriate value of area under the ROC curve (AUC 0.80) to discriminate between the two groups. In conclusion, our study identified a panel of inflammatory related miRNAs differentially expressed between PD patients and healthy controls that highlight key pathophysiological processes and may contribute to improve disease diagnosis.

Necroptosis is a regulated cell death pathway morphologically similar to necrosis that depends on the kinase activity of receptor interacting protein 3 (RIP3) and the subsequent activation of the pseudokinase mixed lineage kinase domain-like protein (MLKL), being also generally dependent on RIP1 kinase activity. Necroptosis can be recruited during pathological conditions, usually following the activation of death receptors under specific cellular contexts. In this regard, necroptosis has been implicated in the pathogenesis of multiple disorders, including acute and chronic neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, and multiple sclerosis. Here, we summarize the molecular mechanisms regulating the induction of necroptosis and downstream effectors of this form of cell death, besides exploring non-necroptotic roles for necroptosis-related proteins that may impact on alternative cell death pathways and inflammatory mechanisms in disease. Finally, we outline the recent evidence implicating necroptosis in neurodegenerative conditions and the emerging therapeutic perspectives targeting necroptosis in these diseases., (© 2020 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is driven by dopaminergic neurodegeneration in the substantia nigra pars compacta (SN) and striatum. Although apoptosis is considered the main neurodegenerative mechanism, other cell death pathways may be involved. In this regard, necroptosis is a regulated form of cell death dependent on receptor interacting protein 3 (RIP3), a protein also implicated in apoptosis and inflammation independently of its pro-necroptotic activity. Here, we explored the role of RIP3 genetic deletion in in vivo and in vitro PD models. Firstly, wild-type (Wt) and RIP3 knockout (RIP3ko) mice were injected intraperitoneally with MPTP (40 mg/kg, i.p.), and sacrificed after either 6 or 30 days. RIP3ko protected from dopaminergic neurodegeneration in the SN of MPTP-injected mice, but this effect was independent of necroptosis. In keeping with this, necrostatin-1s (10 mg/kg/day, i.p.) did not afford full neuroprotection. Moreover, MPTP led to DNA fragmentation, caspase-3 activation, lipid peroxidation and BAX expression in Wt mice, in the absence of caspase-8 cleavage, suggesting intrinsic apoptosis. This was mimicked in primary cortical neuronal cultures exposed to the active MPTP metabolite. RIP3 deficiency in cultured cells and in mouse brain abrogated all phenotypes. Curiously, astrogliosis was increased in the striatum of MPTP-injected Wt mice and further exacerbated in RIP3ko mice. This was accompanied by absence of microgliosis and reposition of glial cell line-derived neurotrophic factor (GDNF) levels in the striata of MPTP-injected RIP3ko mice when compared to MPTP-injected Wt mice, which in turn showed a massive GDNF decrease. RIP3ko primary mixed glial cultures also presented decreased expression of inflammation-related genes upon inflammatory stimulation. These findings hint at possible undescribed non-necroptotic roles for RIP3 in inflammation and MPTP-driven cell death, which can contribute to PD progression.

The spread of microtomography as a tool for visualization of soft tissues has had a significant impact on a better understanding of complex biological systems. This technique allows a detailed three-dimensional quantitative view of the specimen to be obtained, correlating its morphological organization with its function, providing valuable insights on the functionality of the tissue. Regularly overlooked, but of great importance, proper sample mounting and preparation are fundamental for achieving the highest possible image quality even for the high-resolution imaging systems currently under development. Here, a quantitative analysis compares some of the most common sample-mounting strategies used for synchrotron-based X-ray microtomography of soft tissues: alcoholic-immersion, paraffin-embedding and critical-point drying. These three distinct sample-mounting strategies were performed on the same specimen in order to investigate their impact on sample morphology regardless of individual sample variation. In that sense, the alcoholic-immersion strategy, although causing less shrinkage to the tissue, proved to be the most unsuitable approach for a high-throughput high-resolution imaging experiment due to sample drifting. Also, critical-point drying may present some interesting advantages regarding image quality but is also incompatible with a high-throughput experiment. Lastly, paraffin-embedding is shown to be the most suitable strategy for current soft tissue microtomography experiments. Such detailed analysis of biological sample-mounting strategies for synchrotron-based X-ray microtomography are expected to offer valuable insights on the best approach for using this technique for 3D imaging of soft tissues and following morphometric analysis.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Parkin is an E3 ubiquitin ligase involved in Parkinson's disease (PD). Necroptosis is a regulated form of cell death that depends on receptor interacting protein 1 (RIP1) and 3 (RIP3). Importantly, parkin has been implicated in ubiquitination events that can alter inflammation and necroptosis. Here, we investigated how parkin influences microglial function. Incubation of BV-2 microglial cells with zVAD.fmk (zVAD) induced high levels of cell death and viability loss, while N9 microglial cells and primary microglia required further stimuli. Importantly, necrostatin-1 (Nec-1), an inhibitor of RIP1 kinase activity, abrogated cell death, thus implicating RIP1-dependent necroptosis in cell death. Cell death was characterized by necrosome assembly, as determined by sequestration of RIP1/RIP3 in insoluble fractions and by MLKL phosphorylation, which were all abolished by Nec-1. Also, necroptosis-inducing conditions led to TNF-α secretion, which may in turn contribute to autocrine necroptosis activation. Interestingly, parkin knockdown protected BV-2 cells from zVAD-induced necroptosis, which may depend on the higher RIP1 ubiquitination levels detected in siRNA-PARK2 transfected cells. This effect was independent of inflammation, since pro-inflammatory stimulation of BV-2 and primary microglia with silenced parkin resulted in stronger pro-inflammatory gene expression, an opposite observation from zVAD-exposed BV-2 cells. LPS-mediated inflammation was exacerbated by NF-κB/JNK over-activation. Finally, no alterations in mitochondrial ROS production were detected in any condition, thereby excluding the role of parkin in mitophagy. In conclusion, here, we reveal that parkin may have unsuspected roles in microglia by modulating ubiquitination. Parkin loss exacerbates inflammation and promotes survival of activated microglia, thus contributing to chronic neuroinflammation.

The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain.

Necroptosis is a regulated form of necrosis, which may be critical in the pathogenesis of neurodegenerative diseases. Neuroinflammation, characterized by the activation of glial cells such as microglia, is closely linked with neurodegenerative pathways and constitutes a major mechanism of neural damage and disease progression. Importantly, inhibition of necroptosis results in disease improvement, unveiling an alternative approach for therapeutic intervention. In the present study, we screened a small library of new molecules, potentially inhibitors of necroptosis, using two cellular models of necroptosis. A new oxazolone, Oxa12, reduced tumour necrosis factor α (TNF-α)-induced necroptosis in mouse L929 fibrosarcoma cells. Notably, Oxa12 strongly inhibited zVAD-fmk-induced necroptosis in murine BV2 microglial cells. Moreover, Oxa12 blocked phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and interfered with necrosome complex formation, indicating that Oxa12 targets components upstream of MLKL. In fact, in silico molecular docking studies revealed that Oxa12 is occupying a region similar to the 1-aminoisoquinoline type II kinase inhibitor inside the receptor-interacting protein 1 (RIP1) kinase domain. Finally, in microglial cells, Oxa12 attenuated zVAD-fmk- and lipopolysaccharide (LPS)-induced inflammatory processes, as revealed by a marked decrease of TNF-α and/or IL-1β expression. More specifically, Oxa12 negatively targeted c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, as well as NF-κB activation. Overall, we identified a strong lead inhibitor of necroptosis that is also effective at reducing inflammation-associated events. Oxa12 is a promising candidate molecule for further development to target disease states dependent on RIP kinase activity., Competing Interests: The authors declare that they have no conflict of interest.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

We have previously demonstrated the involvement of specific apoptosis-associated microRNAs (miRNAs), including miR-34a, in mouse neural stem cell (NSC) differentiation. In addition, a growing body of evidence points to a critical role for autophagy during neuronal differentiation, as a response-survival mechanism to limit oxidative stress and regulate synaptogenesis associated with this process. The aim of this study was to further investigate the precise role of miR-34a during NSC differentiation. Our results showed that miR-34a expression was markedly downregulated during neurogenesis. Neuronal differentiation and cell morphology, synapse function, and electrophysiological maturation were significantly impaired in miR-34a-overexpressing NSCs. In addition, synaptotagmin 1 (Syt1) and autophagy-related 9a (Atg9a) significantly increased during neurogenesis. Pharmacological inhibition of autophagy impaired both neuronal differentiation and cell morphology. Notably, we showed that Syt1 and Atg9a are miR-34a targets in neural differentiation context, markedly decreasing after miR-34a overexpression. Syt1 overexpression and rapamycin-induced autophagy partially rescued the impairment of neuronal differentiation by miR-34a. In conclusion, our results demonstrate a novel role for miR-34a regulation of NSC differentiation, where miR-34a downregulation and subsequent increase of Syt1 and Atg9a appear to be crucial for neurogenesis progression.

Alzheimer's disease (AD) is a neurodegenerative disorder hallmarked by the accumulation of extracellular amyloid-β (Aβ) peptide and intraneuronal hyperphosphorylated tau, as well as chronic neuroinflammation. Tauroursodeoxycholic acid (TUDCA) is an endogenous anti-apoptotic bile acid with potent neuroprotective properties in several experimental models of AD. We have previously reported the therapeutic efficacy of TUDCA treatment before amyloid plaque deposition in APP/PS1 double-transgenic mice. In the present study, we evaluated the protective effects of TUDCA when administrated after the onset of amyloid pathology. APP/PS1 transgenic mice with 7 months of age were injected intraperitoneally with TUDCA (500 mg/kg) every 3 days for 3 months. TUDCA treatment significantly attenuated Aβ deposition in the brain, with a concomitant decrease in Aβ₁₋₄₀ and Aβ₁₋₄₂ levels. The amyloidogenic processing of amyloid precursor protein was also reduced, indicating that TUDCA interferes with Aβ production. In addition, TUDCA abrogated GSK3β hyperactivity, which is highly implicated in tau hyperphosphorylation and glial activation. This effect was likely dependent on the specific activation of the upstream kinase, Akt. Finally, TUDCA treatment decreased glial activation and reduced proinflammatory cytokine messenger RNA expression, while partially rescuing synaptic loss. Overall, our results suggest that TUDCA is a promising therapeutic strategy not only for prevention but also for treatment of AD after disease onset., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although regarded as neurotoxic, amyloid β (Aβ) peptides may also mediate a wide range of nonpathogenic processes. Autophagy has been implicated in Aβ-mediated effects, although its precise function in neural differentiation remains unknown. Here, we addressed the role of different Aβ fragments in neural stem cell (NSC) proliferation and differentiation, and investigated whether autophagy is involved in Aβ-induced alterations of neural fate. Our results demonstrate that neuronal and glial-specific protein markers are significantly induced by both Aβ1-40 and Aβ1-42. However, Aβ1-40 preferentially enhances neurogenesis of NSCs, as determined by βIII-tubulin, NeuN, and MAP2 neuronal marker immunoreactivity, while Aβ1-42 appears to favor gliogenesis. In contrast, Aβ25-35 does not influence NSC fate. The effect of Aβ1-40 on neurogenesis is partially dependent on its role in NSC self-renewal as both S-phase of the cell cycle and BrdU labeling were markedly increased. Nevertheless, Aβ1-40 resulted also in increased Tuj1 promoter activity. Autophagy, assessed by conversion of endogenous LC3-I/II, fluorescence of pGFP-LC3-transfected cells, and Atg9 protein levels, was evident in both Aβ1-40- and Aβ1-42-treated NSCs, independently of reactive oxygen species production and apoptosis. Finally, inhibition of autophagy by pharmacologic means abrogated Aβ-induced lineage-specific protein markers. These results support distinct roles for different Aβ peptides in NSC fate decision and underline the importance of autophagy control of this process.

p53 plays an important role in regulating a wide variety of cellular processes, such as cell cycle arrest and/or apoptosis. Dysfunction of p53 is frequently associated with several pathologies, such as cancer and neurodegenerative diseases. In recent years substantial progress has been made in developing novel p53-activating molecules. Importantly, modulation of p53 interaction with its main inhibitor, Mdm2, has been highlighted as a promising therapeutic target. In this regard, bimolecular fluorescence complementation (BiFC) analysis, by providing direct visualization of protein interactions in living cells, offers a straightforward method to identify potential modulators of protein interactions. In this study, we developed a simple and robust Venus-based BiFC system to screen for modulators of p53-p53 and p53-Mdm2 interactions in live mammalian cells. We used nutlin-3, a well-known disruptor of p53-Mdm2 interaction, to validate the specificity of the assay. The reduction of BiFC signal mediated by nutlin-3 was correlated with an increase in Puma transactivation, PARP cleavage, and cell death. Finally, this novel BiFC approach was exploited to identify potential modulators of p53-Mdm2 complex formation among a commercially available chemical library of 33 protein phosphatase inhibitors. Our results constitute "proof-of-concept" that this model has strong potential as an alternative to traditional target-based drug discovery strategies. Identification of new modulators of p53-p53 and p53-Mdm2 interactions will be useful to achieve synergistic drug efficacy with currently used anti-tumor therapies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Free-floating right atrial thrombi are rare but associated with high mortality. Although advances in echocardiography have improved diagnosis, their management is still the subject of debate. A 24-year-old woman with a history of smoking, obesity and oral contraceptive use presented to the emergency department with dyspnea, cough and hemoptysis. Transthoracic echocardiography revealed a large free-floating cardiac mass occupying the right atrial chamber and restricting tricuspid valve opening. In view of recurrent pulmonary embolism, she was referred for cardiac surgery and the cardiac mass was excised. Anatomopathological analysis revealed an organized and calcified thrombus. Genetic study showed her to be homozygous for the 4G/4G allelic variant of plasminogen activator inhibitor-1 and heterozygous for the allelic variant A1298C of 5,10-methylenetetrahydrofolate reductase., (Copyright © 2010 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.)

Introduction: Acute myocardial infarction (AMI) in the very elderly is common and is associated with increased mortality. Despite this, the majority of such patients do not receive the most effective cardiovascular therapies. The presence of non-cardiac comorbidities constitutes an additional challenge to the management of AMI in very elderly patients., Objective: To determine the prevalence of non-cardiac comorbidities in the very elderly (age > or = 80 years) with AMI and how it influences their management and in-hospital mortality., Methods: A total of 132 patients consecutively admitted with a diagnosis of AMI from January 2005 to December 2007 were analyzed retrospectively. Two groups were considered: patients with non-cardiac comorbidities (group 1) and those without non-cardiac comorbidities (group 2). Cardiovascular risk factors and non-cardiac comorbidities (anemia, chronic obstructive pulmonary disease, chronic renal failure, cancer, neurologic or psychiatric disorders, and prostatic hyperplasia in men) were recorded. Use of an invasive strategy and the therapy prescribed at discharge were compared between the groups., Results: Non-cardiac comorbidities were found in 56.8% of patients, with the following prevalences: anemia 18.2%; chronic obstructive pulmonary disease 11.4%; chronic renal failure 25.8%; cancer 3.0%; neurologic or psychiatric disorders 11.4%; and prostatic hyperplasia 20.5%. Patients with comorbidities had longer hospital stay than those without (12.1 +/- 5.5 and 10.1 +/- 3.5 days, respectively; p = 0.014). An invasive strategy, with coronary angiography, was used in 12.1% of patients, with no differences between groups (12.3% in patients without comorbidities and 12.0% in those with, p = 0.82). At discharge, more than 70% of the patients were prescribed aspirin, statins and nitrates. With the exception of non-dihydropyridine calcium antagonists, which were more frequently prescribed in patients with comorbidities (15.9% vs. 2.2%; p = 0.04), no other differences in therapy were observed between the two groups., Conclusion: In our population of very elderly patients, the prevalence of non-cardiac comorbidities was high (56.8%), but this did not significantly influence the management of these patients.

Heart failure (HF) is still one of the most important causes of morbidity and mortality worldwide. Neurohormonal changes appear to play an important role in the development and continuation of HF. Among the mediators responsible for these changes, antidiuretic hormone (ADH) is probably the least known. However, elevated concentrations of ADH are frequently found in this syndrome and have prognostic value in addition to known biomarkers. Recent experimental studies and clinical trials have aroused interest in the possible benefits of ADH receptor antagonists. This article reviews the pathophysiological mechanisms of ADH in HF and the latest advances in ADH antagonism in the therapeutic management of HF.