José Fuentes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.

Arginase 1, an enzyme induced by Th2 cytokines, is a hallmark of alternatively activated macrophages and is responsible for the hydrolysis of L-arginine into ornithine, the building block for the production of polyamines. Upregulation of arginase 1 has been observed in a variety of diseases, but the mechanisms by which arginase contributes to pathology are not well understood. We reveal here a unique role for arginase 1 in the pathogenesis of nonhealing leishmaniasis, a prototype Th2 disease, and demonstrate that the activity of this enzyme promotes pathology and uncontrolled growth of Leishmania parasites in vivo. Inhibition of arginase activity during the course of infection has a clear therapeutic effect, as evidenced by markedly reduced pathology and efficient control of parasite replication. Despite the clear amelioration of the disease, this treatment does not alter the Th2 response. To address the underlying mechanisms, the arginase-induced L-arginine catabolism was investigated and the results demonstrate that arginase regulates parasite growth directly by affecting the polyamine synthesis in macrophages.

Abstract Complex regulation of T cell functions during pregnancy is required to ensure materno‐fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate L ‐arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase‐mediated L ‐arginine depletion induces down‐regulation of CD3ζ, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase‐mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous L ‐arginine restored CD3ζ chain expression and T cell proliferation. Thus, L ‐arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.

Mitochondria form close physical contacts with a specialized domain of the endoplasmic reticulum (ER), known as the mitochondria-associated membrane (MAM). This association constitutes a key signaling hub to regulate several fundamental cellular processes. Alterations in ER-mitochondria signaling have pleiotropic effects on a variety of intracellular events resulting in mitochondrial damage, Ca2+ dyshomeostasis, ER stress and defects in lipid metabolism and autophagy. Intriguingly, many of these cellular processes are perturbed in neurodegenerative diseases. Furthermore, increasing evidence highlights that ER-mitochondria signaling contributes to these diseases, including Parkinson's disease (PD). PD is the second most common neurodegenerative disorder, for which effective mechanism-based treatments remain elusive. Several PD-related proteins localize at mitochondria or MAM and have been shown to participate in ER-mitochondria signaling regulation. Likewise, PD-related mutations have been shown to damage this signaling. Could ER-mitochondria associations be the link between pathogenic mechanisms involved in PD, providing a common mechanism? Would this provide a pharmacological target for treating this devastating disease? In this review, we aim to summarize the current knowledge of ER-mitochondria signaling and the recent evidence concerning damage to this signaling in PD.

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases.

Abstract Background Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β‐glucocerebrosidase‐1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S ‐GBA1 mutation on cellular homeostasis and vulnerability in a patient‐specific cellular model of PD. Methods We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy‐lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. Results The N370S mutation produced a significant reduction in β‐glucocerebrosidase‐1 protein and enzyme activity and β‐glucocerebrosidase‐1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Conclusions Our results support a connection between the loss of β‐glucocerebrosidase‐1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1 ‐PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1 ‐PD shares common features with lipid‐storage diseases. © 2017 International Parkinson and Movement Disorder Society

Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1′-dimethyl-4,4′-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP+ (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

Mitochondria-associated membranes (MAMs) are structures that regulate physiological functions between endoplasmic reticulum (ER) and mitochondria in order to maintain calcium signaling and mitochondrial biogenesis. Several proteins located in MAMs, including those encoded by PARK genes and some of neurodegeneration-related proteins (huntingtin, presenilin, etc.), ensure this regulation. In this regard, MAM alteration is associated with neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and Huntington's diseases (HD) and contributes to the appearance of the pathogenesis features, i.e., autophagy dysregulation, mitochondrial dysfunction, oxidative stress, and lately, neuronal death. Moreover,, ER stress and/or damaged mitochondria can be the cause of these disruptions. Therefore, ER-mitochondria contact structure and function are crucial to multiple cellular processes. This review is focused on the molecular interaction between ER and mitochondria indispensable to MAM formation and on MAM alteration-induced etiology of neurodegenerative diseases.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder. While most PD cases are idiopathic, the known genetic causes of PD are useful to understand common disease mechanisms. Recent data suggests that autophagy is regulated by protein acetylation mediated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. The changes in histone acetylation reported to be involved in PD pathogenesis have prompted this investigation of protein acetylation and HAT and HDAC activities in both idiopathic PD and G2019S leucine-rich repeat kinase 2 (LRRK2) cell cultures. Fibroblasts from PD patients (with or without the G2019S LRRK2 mutation) and control subjects were used to assess the different phenotypes between idiopathic and genetic PD. G2019S LRRK2 mutation displays increased mitophagy due to the activation of class III HDACs whereas idiopathic PD exhibits downregulation of clearance of defective mitochondria. This reduction of mitophagy is accompanied by more reactive oxygen species (ROS). In parallel, the acetylation protein levels of idiopathic and genetic individuals are different due to an upregulation in class I and II HDACs. Despite this upregulation, the total HDAC activity is decreased in idiopathic PD and the total HAT activity does not significantly vary. Mitophagy upregulation is beneficial for reducing the ROS-induced harm in genetic PD. The defective mitophagy in idiopathic PD is inherent to the decrease in class III HDACs. Thus, there is an imbalance between total HATs and HDACs activities in idiopathic PD, which increases cell death. The inhibition of HATs in idiopathic PD cells displays a cytoprotective effect.

Lipid and cholesterol metabolism might play a role in the pathogenesis of Parkinson disease (PD). However, the association between cholesterol and PD is not clearly established. Cholesterol accumulation is closely related to the expression of multilamellar bodies (MLBs). Also, cholesterol controls autophagosome transport. Thus, impaired cholesterol and autophagosome trafficking might lead to robust autophagic vacuole accumulation. Our recent work provides the first evidence that the presence of the N370S GBA mutation produces an accumulation of cholesterol, which alters autophagy-lysosome function with the appearance of MLBs, rendering the cell more vulnerable and sensitive to apoptosis.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease. The principal pathological feature of PD is the progressive loss of dopaminergic neurons in the ventral midbrain. This pathology involves several cellular alterations: oxidative stress, mitochondrial dysfunction, loss of proteostasis, and autophagy impairment. Moreover, in recent years, lipid metabolism alterations have become relevant in PD pathogeny. The modification of lipid metabolism has become a possible way to treat the disease. Because of this, we analyzed the effect and possible mechanism of action of linoleic acid (LA) on an SH-SY5Y PD cell line model and a PD mouse model, both induced by 6-hydroxydopamine (6-OHDA) treatment. The results show that LA acts as a potent neuroprotective and anti-inflammatory agent in these PD models. We also observed that LA stimulates the biogenesis of lipid droplets and improves the autophagy/lipophagy flux, which resulted in an antioxidant effect in the in vitro PD model. In summary, we confirmed the neuroprotective effect of LA in vitro and in vivo against PD. We also obtained some clues about the novel neuroprotective mechanism of LA against PD through the regulation of lipid droplet dynamics.

Time series are key across industrial and research areas for their ability to model behaviour across time, making them ideal for a wide range of use cases such as event monitoring, trend prediction or anomaly detection. This is even more so due to the increasing monitoring capabilities in many areas, with the subsequent massive data generation. But it is also interesting to consider the potential of time series for Machine Learning processing, often fused with Big Data, to search for useful information and solve real-world problems. However, time series can be studied individually, representing a single entity or variable to be analysed, or in a grouped fashion, to study and represent a more complex entity or scenario. In this latter case we are dealing with multivariate time series, which usually imply different approaches when dealt with. In this paper, we present a pipeline architecture to process and cluster multiple groups of multivariate time series. To implement this, we apply a multi-process solution composed by a feature-based extraction stage, followed by a dimension reduction, and finally, several clustering algorithms. The pipeline is also highly configurable in terms of the stage techniques to be used, allowing to perform a search with several combinations for the most promising results. The pipeline has been experimentally applied to batches of HPC jobs from different users of a supercomputer, with the multivariate time series coming from the monitoring of several node resource metrics. The results show how it is possible to apply this multi-process information fusion to create different meaningful clusters from the batches, using only the time series, without any labelling information, thus being an unsupervised scenario. Optionally, the pipeline also supports an outlier detection stage to find and separate jobs that are radically different when compared to others on a dataset. These outliers can be removed for a better clustering, and later reviewed looking for anomalies, or if numerous, fed back to the pipeline to identify possible groupings. The results also include some outliers found in the experiments, as well as scenarios where they are clustered, or ignored and not removed at all. In addition, by leveraging Big Data technologies like Spark, the pipeline is proven to be scalable by working with up to hundreds of jobs and thousands of time series.

Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP+ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 microM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.

This paper describes mussel culture in Galicia (N.W. Spain) based on our previous studies and data obtained from a survey carried out in 1984 on 337 rafts (10% of the total) in the Rias of Arousa, Pontevedra and Vigo. Data are also included on the biology of the cultured mussel, raft characteristics and culture techniques. The raft culture process is divided into four stages: obtaining the seed, growing the seed, thinning out the ropes and transferring the seed onto new ropes and final harvest and sale. These operations differ among the different rias and the zone of the ria. Therefore seed can be obtained directly from the coastal rocks or from collectors over the rafts, or both. Seed is attached to ropes that will be thinned out twice or three times before commercial size is reached. Collectors are placed in spring and the mussel seed ropes are placed in the sea throughout the whole year, but specially from November to March. The thinning out process is usually carried out between June and October. The mean surface area of the rafts is 261 m2 in the Ria de Vigo, 352 m2 in the Ria de Pontevedra and 369 m2 in the Ria de Arousa, with an increase in the last of almost 25% since 1974. The declared production per ha and year reaches 48.0 t/raft in the Ria de Arousa, 46.9 t/raft in the Ria de Pontevedra and 33.2 t/raft in the Ria de Vigo, while production per m2 of raft is 130 kg, 133 kg and 127 kg, respectively.

J. Neurochem . (2009) 109 , 889–898. Abstract The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age‐related neurodegenerative disorders, including Parkinson’s disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ‐1 have been reported to be associated with autosomal recessive, early‐onset parkinsonism. In this paper we established a model system to study the involvement of the DJ‐1 protein in paraquat‐induced autophagy. When human neuroblastoma SH‐SY5Y cells were transfected with DJ‐1‐specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat‐induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3‐methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ‐1 in the autophagic response produced by paraquat, providing evidence for the role of PD‐related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH-requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat-mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat-mediated toxicity.

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24 h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3 h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c‐Jun‐N‐terminal kinase (JNK), Akt, and 5′AMP‐activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3–12 hr) ER stress characterized by an increase of Ser51 P‐eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P‐JNK1/2/JNK1/2, Thr172 P‐AMPKα, Ser413 P‐Foxo3a, Thr308 P‐AKt/AKt and Thr32 P‐Foxo3a/Foxo3a ratios, and reduction of Ser2481 P‐mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin‐1, Bcl‐xL, Bcl‐2, autophagy markers, and reduction of myeloid cell leukemia‐1 (Mcl‐1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P‐AKt/AKt and Ser473 P‐AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase‐3 activity (24 hr). Small interfering‐RNA (si‐RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase‐3 in sorafenib‐treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor‐derived xenograft model. In conclusion, the early sorafenib‐induced ER stress and regulation of JNK and AMPK‐dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK‐CHOP‐dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt‐ and AMPK‐related signaling leading to Foxo3a‐dependent BIM EL upregulation.

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.

The research of new biomarkers for Parkinson's disease is essential for accurate and precocious diagnosis, as well as for the discovery of new potential disease mechanisms and drug targets. The main objective of this work was to identify metabolic changes that might serve as biomarkers for the diagnosis of this neurodegenerative disorder. For this, we profiled the plasma metabolome from mice with neurotoxin-induced Parkinson's disease as well as from patients with familial or sporadic Parkinson's disease. By using mass spectrometry technology, we analyzed the complete metabolome from healthy volunteers compared to patients with idiopathic or familial (carrying the G2019S or R1441G mutations in the LRRK2 gene) Parkinson's disease, as well as, from mice treated with 6-hydroxydopamine to induce Parkinson disease. Both human and murine Parkinson was accompanied by an increase in plasma levels of unconjugated bile acids (cholic acid, deoxycholic acid and lithocholic acid) and purine base intermediary metabolites, in particular hypoxanthine. The comprehensive metabolomic analysis of plasma from Parkinsonian patients underscores the importance of bile acids and purine metabolism in the pathophysiology of this disease. Therefore, plasma measurements of certain metabolites related to these pathways might contribute to the diagnosis of Parkinson's Disease.

Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ER stress-induced ASK1 activation could play an important role both in neuronal apoptosis and an autophagic response in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease. The mechanism by which ASK1 executes apoptosis and/or autophagy under ER stress is still unclear. We have addressed this question using SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression with or without PQ. These data support the idea that the autophagic response could have a protector role. We found also an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure. These data suggest that the WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. In conclusion, we report that PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1. However, when the toxic stimuli remain, the cell eventually succumbs to apoptosis.

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD.

A series of aminoketalic castanospermine analogues incorporating a stereoelectronically anchored axial hydroxy group at the pseudoanomeric stereocenter (C-5) have been synthesized to satisfy the need for glucosidase inhibitors that are highly selective for α-glucosidases. The polyhydroxylated bicyclic system was built from readily available hexofuranose derivatives through a synthetic scheme that involved (i) the construction of a five-membered cyclic (thio)carbamate or (thio)urea moiety at the nonreducing end and (ii) the intramolecular nucleophilic addition of the heterocyclic thiocarbamic nitrogen atom to the masked aldehyde group of the monosaccharide. A biological screening of the resulting reducing 2-oxa- and 2-azaindolizidines against several glycosidase enzymes is reported.

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 224:149-156 (2001) - doi:10.3354/meps224149 Two-dimensional gel electrophoresis of Mytilus galloprovincialis: differences in protein expression between intertidal and cultured mussels J. L. López1,*, E. Mosquera1, J. Fuentes2, A. Marina3, J. Vázquez3, G. Alvarez1 1Departamento de Biología Fundamental, Facultad de Biología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain 2Centro de Investigaciones Mariñas, Consellería de Pesca, Xunta de Galicia, 36620 Pontevedra, Spain 3Centro de Biología Molecular Severo Ochoa, CSIC Universidad Autónoma de Madrid, 28049 Madrid, Spain *E-mail: jllopez@usc.es ABSTRACT: Two-dimensional gel electrophoresis (2-DE) is a unique method of large-scale protein characterisation, and is a powerful approach in the study of protein expression. In the present work, the experimental conditions for 2-DE of foot proteins from the mussel Mytilus galloprovincialis Lmk were established; the technique was performed with intertidal and cultured mussels using Melanie 3 software for data analysis. This powerful technique enabled the visualisation of a total of 750 protein spots consistently expressed in the foot. The intensity of 92 selected spots was compared between intertidal and cultured mussels, and statistically significant differences were detected in the expression of 45 (48.9%) of the 92 proteins analysed. In 31 of these proteins, intensity was higher in the cultured stock than in the intertidal mussels, while in 14 proteins spot intensity was higher in the latter. Using mass spectrometry (MS) combined with sequence database searching, 6 of the most prominent differentially expressed proteins were analysed. Of these, 1 was identified as being Heat-shock Protein 70, and 2 were shown to be cytoskeleton-associated proteins, myosin and actin. Heat-shock Protein 70, which is known to be involved in cellular transport and chaperoning and associated with stress situations, was more highly expressed in intertidal mussels living in littoral areas than in cultured mussels. These findings are discussed in connection with the molecular changes involved in the adaptation of mussels to different ecological conditions. KEY WORDS: Mytilus galloprovincialis · Two-dimensional gel electrophoresis · Proteome · Foot proteins Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 224. Online publication date: December 18, 2001 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2001 Inter-Research.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTChiral 2-thioxotetrahydro-1,3-O,N-heterocycles from carbohydrates. 2. Stereocontrolled synthesis of oxazolidine pseudo-C-nucleosides and bicyclic oxazine-2-thionesJose M. Garcia Fernandez, Carmen Ortiz Mellet, and Jose FuentesCite this: J. Org. Chem. 1993, 58, 19, 5192–5199Publication Date (Print):September 1, 1993Publication History Published online1 May 2002Published inissue 1 September 1993https://doi.org/10.1021/jo00071a032RIGHTS & PERMISSIONSArticle Views488Altmetric-Citations58LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Abstract The neuropathology associated with Parkinson's disease (PD) is thought to involve excessive production of free radicals, dopamine autoxidation, defects in glutathione peroxidase expression, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and 1‐methyl‐4‐phenylpyridinium ion (MPP + ), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. We show here that the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) increased significantly after treatment of cultured cerebellar granule cells (CGCs) with 50 μM MPP + . Co‐treatment with antioxidants such as ascorbate (ASC), catalase, α‐tocopherol (α‐TOH), coenzyme Q 10 (CoQ 10 ) or superoxide dismutase (SOD) rescued the cells from MPP + ‐induced death. MPP + ‐induced cell death was also abolished by co‐treatment with nitric oxide synthase (NOS) inhibitors such as 7‐nitroindazole (7‐NI), 2‐ethyl‐2‐thiopseudourea hydrobromide (EPTU) or S ‐methylisothiourea sulphate (MPTU). We also tested the protective effects of an iron chelator (deferoxamine mesylate, DFx) and a peroxynitrite scavenger (FeTTPS) and the results lend further support to the view that the free radical cytotoxicity plays an essential role in MPP + ‐induced death in primary cultures of CGC.

β-d-Gluco and mannopyranosyl selenoureas have been prepared by coupling of the corresponding glycosylamines with phenyl isoselenocyanate in aqueous pyridine. Alkyl and aryl isoselenocyanates, and 1,4-phenylene diisoselenocyanate have been obtained from the corresponding formamides with an excess of triphosgene, black selenium and triethylamine. Treatment of the O-unprotected β-d-glucopyranosyl selenourea with aqueous oxygen peroxide afforded a 1,2-trans-fused bicyclic isourea.

The relative influence of three factors on the growth rate of mussels, Mytilus galloprovincialis, cultivated in an inner area of the Rı́a de Arousa (NW Spain), was analysed. The three factors acted within the raft: position of the culture ropes on the raft, depth of cultivation and stocking density. Results indicate that depth of cultivation is the major factor affecting the growth of the mussels. In the two phases of the cultivation process, from seeding to thinning out and from thinning out to harvest, mussels cultivated in the upper part of the water column (2.5 m depth) were significantly longer and heavier than those in the lower part (7.5 m depth). Effect of the position of the ropes on the raft was less important. Only at harvest, were significant differences in the weight of the mussels detected, with lighter mussels occurring at the back (down-current) of the raft. Surprisingly, stocking density showed no significant effect on the growth of the mussels in any of the two cultivation phases. From the results obtained in this experiment, some recommendations, important for the management of the Galician mussel cultivation, are given.

PRIMARY cultures of cerebellar granule neurons, maintained in a serum-containing medium, underwent apoptosis when exposed to C2-ceramide, as assessed by mitochondrial reduction of MTT and intranucleosomal DNA fragmentation. After an 8h exposure to 50 μM C2-ceramide, cell viability decreased by 25–40%. Addition of lithium together with C2-ceramide resulted in a partial protection of apoptosis, which was maximal ab mM lithium (37% protection). When lithium was added h before the apoptotic stimulus the neuroprotective effect of the ion was clearly increased (66% protection). This effect was not due to intracellular inositol depletion or inhibition of NMDA receptors. Our data broaden the nature of apoptotic insults being reversed by lithium, stressing the neuroprotective effects of the ion.

In this article, the growth, mortality, pathological conditions and protein expression of hatchery obtained mussels from pure and hybrid crosses between individuals from three genetically divergent European populations were evaluated in the Rı́a de Arousa (NW Spain) under raft-suspended cultivation conditions. Progenitors for the crosses were obtained from a Mytilus edulis population from The Netherlands and from two M. galloprovincialis populations located on each of the two sides of a major genetic break, associated with the Almerı́a–Oran oceanographic front in the Iberian Peninsula. Results indicated that mussels from crosses between individuals of M. galloprovincialis populations have a significantly higher biomass production than those from hybrid crosses between individuals of M. galloprovincialis and M. edulis populations. This different performance was not due to differences in growth rate but rather to the considerably higher mortality, during the warmer season, of the mussels from hybrid crosses. This strong viability selection operating against hybrid mussels with respect to pure M. galloprovincialis crosses under raft cultivation conditions in the Rı́a de Arousa was very similar to that operating in natural populations of the M. edulis/M. galloprovincialis hybrid zone in SW England, reported in previous studies. Our results also show that the lower viability of hybrid mussels is clearly associated with both higher parasitization by the protistan Marteilia refringens and lower levels of the stress proteins calreticulin and heat shock protein 70. Among the mussels from the different M. galloprovincialis crosses, those from crosses between males and females of autochthonous (Galician) origin are the ones which show a better performance.

Bicyclic azasugar glycomimetics structurally related to the polyhydroxy-indolizine and -piperidine series incorporating a stereoelectronically controlled pseudoanomeric axial hydroxy group have been prepared by tautomeric rearrangement of cyclic thiocarbamate precursors; preliminary enzyme inhibition tests show an increased selectivity towards yeast α-glucosidase for the α-D-glucopyranose analogue as compared with castanospermine or nojirimicin.

Exposure of cerebellar granule cells (CGCs) to 1-methyl-4-phenylpyridinium (MPP+) results in apoptotic cell death, which is markedly attenuated by co-treatment of CGCs with the radical scavenger vitamin E. Analysis of free radical production and mitochondrial transmembrane potential (DeltaPsim), using specific fluorescent probes, showed that MPP+ mediates early radical oxygen species (ROS) production without a loss of DeltaPsim. Exposure to MPP+ also produces an early increase in Bad dephosphorylation and translocation of Bax to the mitochondria. These events are accompanied by cytochrome c release from mitochondria to cytosol, which is followed by caspase 3 activation. Exposure of the neurons to vitamin E maintains Bad phosphorylation and attenuates Bax translocation, inhibiting cytochrome c release and caspase activation. MPP+-mediated cytochrome c release is also prevented by allopurinol, suggesting the participation of xanthine oxidase in the process. Our results indicate that free radicals play an active role in the MPP+-induced early events that culminate with cell death.

A research project to compare productive traits (growth and mortality), disease susceptibility and immune capability between Ostrea edulis stocks was performed. This article reports the results on the immune capability and its relation with infection by the intrahaemocytic protozoan Bonamia ostreae. Four to five oyster spat families were produced from each of four European flat oyster populations (one from Ireland, one from Greece and two from Galicia, Spain) in a hatchery. The spat were transferred to a raft in the Ría de Arousa (Galicia) for on growing for 2 years. Total haemocyte count (THC) and differential haemocyte count (DHC) were estimated monthly through the second year of growing-out. Three types of haemocytes were distinguished: granulocytes (GH), large hyalinocytes (LHH) and small hyalinocytes (SHH). Significant correlations between the mean relative abundance of GH and SHH of the families and the mean prevalence of B. ostreae, the overall incidence of pathological conditions and the cumulative mortality of the families were found; these correlations supported the hypothesis that high %GH and low %SHH would enhance oyster immune ability and, consequently, would contribute to lower susceptibility to disease and longer lifespan. Infection by B. ostreae involved a significant increase of circulating haemocytes, which affected more markedly the LHH type. The higher the infection intensity the higher the %LHH. This illustrates the ability of B. ostreae to modulate the immune responses of the O. edulis to favour its own multiplication. A significant reduction of the phenoloxidase activity in the haemolymph of oysters O. edulis infected by B. ostreae was observed. Nineteen enzymatic activities in the haemolymph of O. edulis and Crassostrea gigas (used as a B. ostreae resistant reference) were measured using the kit api ZYM®, Biomerieux. Qualitative and quantitative differences in enzyme activities in both haemocyte and plasma fractions between B. ostreae noninfected O. edulis from different origins were recorded. However, no clear positive association between enzyme activity and susceptibility to bonamiosis was found. The only enzyme detected in the resistant species C. gigas that was not found in the susceptible one O. edulis was β-glucosidase (in plasma). B. ostreae infected O. edulis showed significant increase of some enzyme activities and the occurrence of enzymes that were not detected in noninfected oysters. These changes could be due to infection-induced enzyme synthesis by the host or to enzyme synthesis by the parasite.

The leishmaniases are a group of vector-borne parasitic diseases that represent a major international public health problem; they belong to the most neglected tropical diseases and have one of the highest rates of morbidity and mortality. The clinical outcome of infection with Leishmania parasites depends on a variety of factors such as parasite species, vector-derived products, genetics, behaviour, and nutrition. The age of the infected individuals also appears to be critical, as a significant proportion of clinical cases occur in children; this age-related higher prevalence of disease is most remarkable in visceral leishmaniasis. The mechanisms resulting in this higher incidence of clinical disease in children are poorly understood. We have recently revealed that sustained arginase activity promotes uncontrolled parasite growth and pathology in vivo. Here, we tested the hypothesis that arginase-mediated L-arginine metabolism differs with age.The age distribution of patients with visceral or cutaneous leishmaniasis was determined in cohorts of patients in our clinics in endemic areas in Ethiopia. To exclude factors that are difficult to control in patients, we assessed the impact of ageing on the manifestations of experimental leishmaniasis. We determined parasite burden, T cell responses, and macrophage effector functions in young and aged mice during the course of infection.Our results show that younger mice develop exacerbated lesion pathology and higher parasite burdens than aged mice. This aggravated disease development in younger individuals does not correlate with a change in T helper cytokine profile. To address the underlying mechanisms responsible for the more severe infections in younger mice, we investigated macrophage effector functions. Our results show that macrophages from younger mice do not have an impaired capacity to kill parasites; however, they express significantly higher levels of arginase 1 than aged mice and promote parasite growth more efficiently. Thus, our results demonstrate that ageing differentially impacts on L-arginine metabolism and subsequent effector functions of physiologically distinct macrophage subsets.Here, we show that arginase-mediated L-arginine metabolism is modulated with age and affects the capacity of macrophages to express arginase; the increased capacity to upregulate this enzyme in younger individuals results in a more permissive environment for parasite growth, increased disease severity and pathology. These results suggest that the difference in arginase-mediated L-arginine catabolism is likely to be an important factor contributing to the increased incidence of clinical cases in children. Thus, targeting L-arginine metabolism might be a promising therapeutic strategy against leishmaniasis, especially in children and young adults.

An experiment was carried out to compare the performance of various cohorts of oysters Ostrea edulis from each of four different geographic origins that were born and cultured in the same environment. This paper reports the variation of gonad condition of the oysters through on growing and analyses of the relationship between gametogenesis and infection by Bonamia ostreae. Nineteen cohorts of spat were produced in a hatchery from four oyster populations: Irish, Greek, and two Galician: Ortigueira and Coroso; the spat were transferred to a raft in the Ría de Arousa, an area affected by bonamiosis, for grow-out for two years. Significant differences were observed in the temporal pattern of gonad condition between oysters from different geographic origins. Irish oysters showed a short period of gonad activity and a long gonad resting phase. By contrast, Greek oysters had the longest period of gonad activity and the shortest gonad resting phase, as well as the highest percentage of male oysters compared to oysters from other origins. Galician oysters, from both origins, showed a pattern of gonad development intermediate between Irish and Greek, with a higher percentage of females during the second reproductive period. Significant differences were also observed between cohorts within Greek, Coroso and Irish origin, but not from Ortigueira, whose cohorts showed a similar temporal pattern of gonad condition. The differences between oysters from different origins would indicate adaptation to different environments; nevertheless the significant differences that were found between cohorts within origins would indicate that there is variability that eventually could allow adaptation to new environments. Considering sex categories, heavy infections by B. ostreae were significantly more frequent in oysters with a predominant female component; considering gonad stage, heavy infections were significantly more frequent in ripe and partially spawned oysters. The results suggest a hypothetical enhancement of progression of infection by B. ostreae associated with female maturation.

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work.

Abstract Background Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. Results The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb ( F. auricularia ) and 0.94 Gb ( E. annulipes ) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. Conclusions This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.

Repeat expansion diseases are a group of neuromuscular and neurodegenerative disorders characterized by expansions of several successive repeated DNA sequences. Currently, more than 50 repeat expansion diseases have been described. These disorders involve diverse pathogenic mechanisms, including loss-of-function mechanisms, toxicity associated with repeat RNA, or repeat-associated non-ATG (RAN) products, resulting in impairments of cellular processes and damaged organelles. Mitochondria, double membrane organelles, play a crucial role in cell energy production, metabolic processes, calcium regulation, redox balance, and apoptosis regulation. Its dysfunction has been implicated in the pathogenesis of repeat expansion diseases. In this review, we provide an overview of the signaling pathways or proteins involved in mitochondrial functioning described in these disorders. The focus of this review will be on the analysis of published data related to three representative repeat expansion diseases: Huntington’s disease, C9orf72-frontotemporal dementia/amyotrophic lateral sclerosis, and myotonic dystrophy type 1. We will discuss the common effects observed in all three repeat expansion disorders and their differences. Additionally, we will address the current gaps in knowledge and propose possible new lines of research. Importantly, this group of disorders exhibit alterations in mitochondrial dynamics and biogenesis, with specific proteins involved in these processes having been identified. Understanding the underlying mechanisms of mitochondrial alterations in these disorders can potentially lead to the development of neuroprotective strategies.

Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.

Paraquat (PQ) (1, 1’-dimethyl-4, 4’-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson’s disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.Addendum toInhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic Cell Death in Neuroblastoma SH-SY5Y CellsR.A. González-Polo, M. Niso-Santano, M.A. Ortíz-Ortíz, A. Gómez-Martín, J.M. Morán, L. García-Rubio, J. Francisco-Morcillo, C. Zaragoza , G. Soler and J.M. FuentesToxicological Science 2007; In press

Curcumin, the active compound of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. This agent has been shown to regulate numerous transcription factors, cytokines, protein kinases, adhesion molecules, redox status and enzymes that have been linked to inflammation. While curcumin has been identified as an activator of apoptosis in several cell lines, the mechanism by which it initiates apoptosis, however, remains poorly understood. We considered curcumin from the point of view of its ability to protect against oxidative stress, the latter being one factor strongly implicated in the development of Parkinson's disease. Although the etiology of Parkinson's disease remains unknown, epidemiological studies have linked exposure to pesticides such paraquat to an increased risk of developing the condition. Analysis of the neurotoxic properties of these pesticide compounds has been focused on their ability to induce oxidative stress in neural cells. Given curcumin's capacity to protect against oxidative stress, it has been considered as a potential therapeutic agent for neurodegenerative diseases such as Parkinson's disease that involve an oxidative stress component. In the present report we describe the effect of curcumin in paraquat-mediated apoptosis of N27 mesencepahlic cells. We show that subtoxic concentrations of curcumin sensitize N27 mesencephalic cells to paraquat-mediated apoptosis.

Abstract Paraquat (PQ) (1,1-dimethyl-4,4′-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). In this sense, understanding of the molecular mechanism underlying PQ-induced toxicity to neural cells is important for optimal use as well as for the development of new drugs. To gain insights into PQ-induced neurotoxicity, polymerase chain reaction (PCR) array analysis focused on a panel of apoptosis-related genes was performed using neuroblastoma SH-SY5Y cells. Up to 65 apoptosis-related genes were monitored. Our analysis of apoptotic process through microarray technology showed that in PQ-induced neuroblastoma SH-SY5Y cells, there is a different expression of BIK, CASP3, CASP7, CRADD, DAPK, FAS, and other related genes, in comparison to unstimulated cells. Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases as PD. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in PQ-mediated toxicity of neural cells. The authors would like to thank to Maria Pura Delgado for helpful assistance in the laboratory work. Miguel Angel Ortiz Ortiz is a predoctoral recipient of a fellowship from Second Vice-presidency and Regional Ministry of Economy, Commerce and Innovation, from the Junta of Extremadura. Rosa Ana González Polo is currently recipient of a “Juan de la Cierva” grant from Spanish Ministry of Education and Science. This work was supported by grants PRI06B124 from Junta de Extremadura and PI040828 and PI070400 from FIS‐ISCIII, Spanish Ministry of Health and Consumer Affairs.

Paraquat (PQ) is a well-known herbicide that exerts its effects by elevating intracellular levels of superoxide. It has been previously demonstrated that oxidative and nitrosative stress participate to PQ-induced cell death. Here, we document that PQ increases the levels of nitric oxide (NO) in rat mesencephalic cells and causes nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to activate the NO/GAPDH/Siah cell death cascade. PQ exposure increases expression of the p300/CREB-binding protein (p300/CBP) and phosphorylation of p53 at Ser 15, which stimulates p53-dependent transactivation through increased binding with p300. Although this cascade could be inhibited by preincubation with the monoamine oxidase B inhibitor deprenyl, cell death was not prevented. Pretreatment of cells with the neuronal nitric oxide synthase inhibitor 7-nitroindazole efficiently prevented the activation of the GAPDH/NO/Siah cell death cascade, thereby protecting cells against PQ-induced toxicity. The results suggest that PQ induces this novel cell death cascade in rat mesencephalic cells, but inhibition of the pathway does not impede cell death because of an oxidative burst generated by the pesticide.

We evaluated differences in productive traits and disease susceptibility among Ostrea edulis stocks. We produced 4 to 5 families from each of 4 oyster populations (Irish, Greek and 2 Galician) in a hatchery. Spat corresponding to 19 different families were transferred to a raft in the Ría de Arousa (Galicia, Spain) for grow-out. Samples of each family were histologically processed every month for 2 yr. One of the pathological conditions disclosed by histological examination was characterised by the occurrence of numerous abnormal cells throughout the connective tissue of various organs, showing hypertrophied nuclei with marginated chromatin and a characteristic large intranuclear acidophilic inclusion. Ultrastructural examination showed that the abnormal cells contained herpesvirus-like particles. In situ hybridisation assay using a DNA probe specific for Ostreid herpesvirus 1 (OsHV-1) confirmed that the abnormal cells were infected by OsHV-1 or a closely related herpesvirus. All cases of this pathological condition, except one, were detected during the first year of grow-out; thus it was mostly restricted to juvenile stages. The disease was detected in oysters of each origin but it was not found in all families of each origin, thus suggesting significant parental influence in the susceptibility to this disease or significant influence of the infective status of the parents on the infection of the progeny (vertical transmission). This pathological condition was likely responsible for oyster mortality to some extent during the first year of grow-out.

Turmeric (curry powder), an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound known as curcumin or diferuloylmethane. Curcumin is a widely studied phytochemical with a variety of biological activities. In addition to its anti-inflammatory and antimicrobial/antiviral properties, curcumin is considered as a cancer chemopreventive agent as well as a modulator of gene expression and a potent antioxidant. Since oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease (PD), curcumin has been proposed to have potential therapeutic value for the treatment of neurodegenerative diseases such as PD. Following age, a family history of PD is the most commonly reported risk factor, suggesting a genetic component of the disease in a subgroup of patients. The LRRK2 gene has emerged as the gene most commonly associated with both familial and sporadic PD. Here, we report that exposure of rat mesencephalic cells to curcumin induces the expression of LRRK2 mRNA and protein in a time-dependent manner. The expression of other PD-related genes, such alpha-synuclein and parkin, was not affected by exposure to curcumin, and PTEN-induced putative kinase 1 (PINK1) was not expressed in rat mesencephalic cells. As LRRK2 overexpression is strongly associated with the pathological inclusions found in several neurodegenerative disorders, further studies are needed to evaluate the effects of curcumin as a therapeutic agent for neurodegenerative diseases.

The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway.

To date, European mussel culture has relied entirely on wild seed from suspended collectors or mussel beds. One problem faced by blue mussel producers is the unpredictability of seed supply, the amounts of wild seed available being extremely variable from year to year. A second problem is that recently spawned mussels cannot be sold due to insufficient meat. Hatcheries can complement wild seed supply. Hatcheries also allow triploid induction that produces non-maturing mussels. In this chapter, the different steps in hatchery production of mussel seed are described. A final section addresses future trends.

Mutations in LRRK2 (leucine-rich repeat kinase 2) are found associated with both sporadic and familial Parkinson's disease (PD). Pathogenic mutations are localized to the catalytic domains of LRRK2, including kinase and GTPase domains. Altered catalytic activity correlates with neurotoxicity, indicating that targeting those activities may provide clues as to novel therapeutic strategies for LRRK2-linked PD. However, the cellular readout of such altered catalytic activities remains largely unknown. Recent cell biological studies have started to highlight possible early cellular events which are altered in the presence of pathogenic LRRK2 and may ultimately lead to neuronal demise, and these studies link altered LRRK2 function to various abnormal endolysosomal vesicular trafficking events. This review examines our current knowledge of LRRK2 neurobiology and how pathogenic mutations may lead to neurodegeneration in PD.

Phenolic compounds derived from olive oil have beneficial health properties against cancer, neurodegenerative, and metabolic diseases. Therefore, there are discrepancies in their impact on mitochondrial function that result in changes in oxidative capacity, mitochondrial respiration, and energetic demands. This review focuses on the versatile role of oleuropein, a potent antioxidant that regulates the AMPK/SIRT1/mTOR pathway to modulate autophagy/mitophagy and maintain metabolic homeostasis.

Readily derived from d-glucose, 5-[(2R,3S,4R)-3,4-dihydroxypyrrolidin-2-yl]-2-methyl-3-furoic esters and amides are selective and competitive inhibitors (Ki≥3 μM) of α-l-fucosidase from bovine epididymis and from human placenta.

A stereocontrolled synthesis of pyranoid and furanoid spironucleosides and spiro-C-glycosides (d-ribo and d-arabino configurations) of oxazolidines, oxazolines and perhydrooxazines via isothiocyanato sugar derivatives is reported. The intermediate isothiocyanates are prepared from sugar spiroketals by stereoselective opening of the acetal ring with trimethylsilyl N- and C-nucleophiles, and later formation of the isothiocyanato group.

Abstract The annual pattern and the spatial variability of settlement of the mussel, Mytilus galloprovincialis , on collectors suspended from rafts, have been investigated in the Ria de Arousa (NW of Spain). A period of settlement, from March to September, was observed at the three sites and three depths investigated. Settlement showed a strong spatial variability with regard to both the situation of the collectors within the Ria and the depth to which they were suspended. The more seaward the site in the Ria and shallower the depth in the water column, the higher the density of settlement.

A new stereocontrolled and high yielding synthesis of biologically active polyhydroxyperhydroazepines is reported starting from easily available glycosylenamines (D-gluco, D-manno, and D-galacto configurations), which are transformed into 1,6-azaanhydropyranose derivatives. O- and N-Deprotection of the latter, followed by reduction with sodium cyanoborohydride, gives the target chiral iminocyclitols. The method is based on the capacity of the dialkoxycarbonylvinyl group to stabilize an amide ion, and the only limitation is the necessity for the starting glycosylenamine to have β-D-configuration. The inhibitory activity of several intermediate iminocyclitols and aldopyranosylenamines on different α- and β-glycosidases is also reported.

A new route for the preparation of four new indolizidines, (1R,2S,6S,7S,8aS)- and (1R,2S,6R,7R,8aS)-1,2,6,7-tetrahydroxyindolizidine (30 and 32) and (1S,2R,7S,8S,8aR)- and (1S,2R,7R,8R,8aR)-1,2,7,8-tetrahydroxyindolizidine (44 and 46), is reported. The synthesis is based on Knoevenagel homologation of the readily available enantiomerically pure pyrrolidin-carbaldehydes 13 and 37followed by asymmetric dihydroxylation of the subsequent alkenyl pyrrolidines and cyclization of the corresponding imino-octitols. The new indolizidines and their precursors (imino-octitols 20, 25, 26) and indolizidinones 28a and 28b have been tested for inhibitory activities toward 26 glycosidases. The enzymatic inhibition of trans-7-hydroxy-d-(-)-swainsonine (44) toward alpha-mannosidases is similar to that described for trans-7-hydroxy-l-(+)-swainsonine (11b) toward naringinase (alpha-l-rhamnosidase from Penicillium decumbens).

The syntheses of 2,3,4-tri-O-acetyl(benzoyl)-β-D-glucopyranosyl isothiocyanates (3, 4) from the corresponding acylated N-(2,2-diethoxycarbonylvinyl)-β-D-glucopyranosylamine are described. Reactions of 3 and 4 with phenacylamine hydrochloride yielded N-phenacyl-N′-(2,3,4-tri-O-acyl-β-D-glucopyranosyl)thioureas (6, 7) whereas treatments of the same compounds with aminoacetone hydrochloride gave the N-nucleoside analogues 5-methyl-1-(2′,3′,4′-tri-O-acyl-β-D-glucopyranosyl)-4-imidazoline-2-thiones (8, 9). The partially protected N-(2-thiazoline-2-yl)-β-D-glucopyranosylamine (10) and the thioureylenedisaccharide II were prepared from 4 and 2-chloroethylamine hydrochloride under different reaction conditions.

The European flat oyster Ostrea edulis is highly susceptible to infection by the protozoan Bonamia ostreae, an intracellular parasite able to survive and proliferate within the oyster haemocytes. On the contrary, the Pacific cupped oyster Crassotrea gigas is resistant. Two-dimensional electrophoresis was adapted to analyse the proteins of the oyster haemolymph. Comparisons of the haemolymph protein profiles between the two oyster species and between O. edulis stocks with different susceptibility to bonamiosis were performed. Differences in the number of proteins detected from each oyster species/stock were recorded; the highest number corresponded to the species resistant to bonamiosis, whereas the lowest number corresponded to the most susceptible O. edulis stock. Protein spots exclusive to each species and stock were detected; identification of those proteins could help to understand the key of tolerance/resistance to bonamiosis. Comparison between “healthy” and B. ostreae infected oysters was also performed. A substantial reduction of the number of proteins in the oyster haemolymph associated with B. ostreae infection was recorded. Protein spots exclusive to healthy and infected groups were detected; identification of haemolymph proteins whose synthesis is induced, repressed, up regulated or down regulated should help to understand the inability of O. edulis to neutralise or overcome B. ostreae infection. The results support a promising utility of 2-D electrophoresis, applied to the analysis of haemolymph proteins, to understand the oyster–B. ostreae interaction and to find the bases of tolerance/resistance to bonamiosis.

Paraquat is a cationic herbicide that causes acute cell injury by undergoing redox cycling. Oxidative stress is thought to be the crucial mechanism invoked by this redox-cycling compound. The cytotoxicity of paraquat was examined in an immortalized human mesencephalic neuron-derived cell line. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction activity was examined as cytotoxicity indicator. Cells were seeded with densities at inoculation of 5 × 10(4)cells/ml and 10 × 10(4)cells/ml, and paraquat was added 24h later to give final concentrations from 10 to 500 μM. At 24 and 48 h of treatment, mitochondrial activity was determined with the MTT assay. To further understand the effect of paraquat exposure on human mesencephalic neuron-derived cells, the cells were differentiated and similar experiments were carried out. Supplementation of culture medium with dibutyryl cyclic AMP and GDNF significantly increased the resistance of the cultures to the paraquat-mediated cytotoxicity. These results confirm that GDNF confers protection against paraquat-mediated cytotoxicity and show that immortalized human mesencephalic neuron-derived cells are an adequate in vitro system for evaluating the cytoprotective effects of GDNF on oxidative injury caused by xenobiotics.

Current evidence shows that numerous classic vascular risk factors (VRF) contribute to mild cognitive impairment (MCI), but the effects of emerging VRFs are less well-known. Using a comprehensive approach, we assessed the frequency and strength of association between MCI and classic VRFs, subclinical markers of atherosclerosis (cystatin C, lipoprotein(a), high-sensitivity C-reactive protein, and intima-media thickness) and white matter hyperintensities (WMH).In this case-control study of consecutive MCI patients and cognitively normal controls, subjects underwent clinical and neuropsychological examinations, laboratory analyses, a carotid duplex scan, and a brain magnetic resonance imaging scan.The study included 105 patients with amnestic MCI (aMCI): 24 with single domain amnestic MCI, 81 with multiple domain amnestic MCI, and 76 controls. Compared to controls, patients with aMCI were significantly older and had higher rates of arterial hypertension, atrial fibrillation, and depression. They also had a larger intima-media thickness and higher load of WMHs, both periventricular (WMHpv) and subcortical (WMHsc). In the adjusted analysis, all variables except WMHsc displayed a significant association with aMCI. Body mass index exerted a protective effect.Our findings suggest a direct association between aMCI and age, hypertension, atrial fibrillation depression, intima-media thickness, and WMHpv. Body mass index has a protective effect on this MCI subtype.

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. This neuronal loss, inherent to age, is related to exposure to environmental toxins and/or a genetic predisposition. PD-induced cell death has been studied thoroughly, but its characterization remains elusive. To date, several types of cell death, including apoptosis, autophagy-induced cell death, and necrosis, have been implicated in PD progression. In this study, we evaluated necroptosis, which is a programmed type of necrosis, in primary fibroblasts from PD patients with and without the G2019S leucine-rich repeat kinase 2 (LRRK2) mutation and in rotenone-treated cells (SH-SY5Y and fibroblasts). The results showed that programmed necrosis was not activated in the cells of PD patients, but it was activated in cells exposed to rotenone. Necrostatin-1 (Nec-1), an inhibitor of the necroptosis pathway, prevented rotenone-induced necroptosis in PD models. However, Nec-1 affected mitochondrial morphology and failed to protect mitochondria against rotenone toxicity. Therefore, despite the inhibition of rotenone-mediated necroptosis, PD models were susceptible to the effects of both Nec-1 and rotenone.

An efficient synthesis of macrocyclic ligands incorporating two α,α′-trehalose subunits linked through the primary C-6,6′ positions by means of 1,3-thiourea spacers is reported; the Z/E configuration of the N–C(S) bonds is governed by intramolecular hydrogen bonding as well as steric factors.

The influence of stock and culture site on growth rate and mortality was investigated in populations of Mytilus galloprovincialis Lmk. cultivated in the area of greatest mussel production in the world (Galicia, N.W. Spain). The experimental results indicate that variations in mortality, from May to August, of mussel seed are mostly determined by the populations selected as source of seed. On the other hand, differences in growth rate over this 3-month period are mainly explained by two components of spatial variation; a site component (variation among rafts) and a micro-site component (variation within raft). In addition, differences in growth rate between the fore and the aft part of the rafts (micro-site component) were clearly higher than differences among rafts placed in distinct growing sites (site component). These results are discussed in connection with the role of both the selection of the source of seed and the management of rafts in the improvement of mussel aquaculture in Spain.

The preparation of sugar ureas and thioureas by nucleophilic addition of water or hydrogen sulfide, respectively, to sugar-derived carbodiimides has been examined. Acetic acid efficiently catalysed the formation of ureas, whereas silica gel was found to be a more convenient catalyst in the case of the thioxo analogues. The procedures have been exploited in the development of an amine- and isocyanate-free synthesis of urea- and thiourea-tethered pseudooligosaccharides via the corresponding glycosylcarbodiimido sugars. The fully unprotected compounds adopted, preferentially, the (Z,Z) configuration at the pseudoamide bonds in water solution.

Cyclic sulfates of N- and C-nucleosides (d-ribo and d-erythro configurations, respectively) are used to prepare 3‘-fluoro and 3‘-azido d-xylo N-nucleosides and l-threo C-nucleosides. The reduction of the 3‘-azido C-nucleosides (furan, imidazoline-2-thione, and pyrrole derivatives) gives 3‘-amino C-nucleosides, which, by reaction with thiocarbonyldiimidazole, are transformed into 3‘-isothiocyanato C-nucleosides. Reaction of the 3‘-amino with the 3‘-isothiocyanato C-nucleosides gives thioureylene di-C-nucleosides, a type of nucleotide analogue with a nonionic bridge isosteric of the phosphate group.

Practical, facile and high-yielding one-pot syntheses of different O-unprotected glycopyranosyl thioureas and thioureido bolaamphiphiles (two-step synthesis) and of 2-amino-4,5-dihydro-(1,2-dideoxy-β-d-glucopyranoso)[1,2-d]oxazoles (three-step synthesis) from glycopyranosylamines are reported. The method involves the preparation of O-unprotected β-d-gluco (and d-galacto)pyranosyl isothiocyanates which are in equilibrium with the corresponding 1,2-cyclic thiocarbamates, coupling with amines to afford glycosyl thioureas and treatment with yellow mercury (II) oxide to give trans-fused bicyclic isoureas. A d-gluco trehazolin analogue is prepared in this way. In situ transformation of N,N-dialkyl, N′-glucopyranosyl thioureas into the corresponding ureas is also reported.

Evidence of the effect of vascular risk factors and white matter lesions on the progression of mild cognitive impairment (MCI) to dementia is not conclusive.The study aimed to analyze the impact of these factors on MCI progression to dementia from a global perspective.Our study included a population of 105 patients with MCI.After a mean follow-up period of 3.09 years (range, 2-3.79), 47 patients (44.76%) progressed to dementia: 32 (30.8%) to mixed dementia, 13 (12.5%) to probable AD, and 2 (1.9%) to vascular dementia. Total cholesterol levels (OR: 1.015 [1.003-1.028]) and LDL cholesterol levels (OR: 1.018 [1.004-1.032]) increased the risk of progression to dementia. Cystatin C was a protective factor against progression to dementia (OR: 0.119 [0.015-0.944], p = 0.044). During the second year of follow-up, the presence of subcortical white matter hyperintensities increased the risk of progression to dementia (OR: 5.854 [1.008- 33.846]). Subcortical and periventricular white matter hyperintensities were also associated with an increased risk of progression to dementia during the second year of follow-up (OR: 3.130 [1.098-8.922] and OR: 3.561 [1.227-10.334], respectively). The same was true for silent infarcts (OR: 4.308 [1.480- 12.500]).A high percentage of patients progressed to dementia. Total cholesterol, LDL cholesterol, and white matter hyperintensities were found to be associated with MCI progression to dementia. In contrast, cystatin C was shown to be a protective factor against progression to dementia.

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD) and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

The identification of Parkinson's disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S-LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder.

Efficient and intuitive Human-Robot interfaces are crucial for expanding the user base of operators and enabling new applications in critical areas such as precision agriculture, automated construction, rehabilitation, and environmental monitoring. In this paper, we investigate the design of human-robot interfaces for the teleoperation of dynamical systems. The proposed framework seeks to find an optimal interface that complies with key concepts such as user comfort, efficiency, continuity, and consistency. As a proof-of-concept, we introduce an innovative approach to teleoperating underwater vehicles, allowing the translation between human body movements into vehicle control commands. This method eliminates the need for divers to work in harsh underwater environments while taking into account comfort and communication constraints. We conducted a study with human subjects using a head-mounted display attached to a smartphone to control a simulated ROV. Also, numerical experiments have demonstrated that the optimal translation is often the most intuitive and natural one, aligning with users' expectations.

Lysosomes are the main organelles responsible for the degradation of macromolecules in eukaryotic cells. Beyond their fundamental role in degradation, lysosomes are involved in different physiological processes such as autophagy, nutrient sensing, and intracellular signaling. In some circumstances, lysosomal abnormalities underlie several human pathologies with different etiologies known as known as lysosomal storage disorders (LSDs). These disorders can result from deficiencies in primary lysosomal enzymes, dysfunction of lysosomal enzyme activators, alterations in modifiers that impact lysosomal function, or changes in membrane-associated proteins, among other factors. The clinical phenotype observed in affected patients hinges on the type and location of the accumulating substrate, influenced by genetic mutations and residual enzyme activity. In this context, the scientific community is dedicated to exploring potential therapeutic approaches, striving not only to extend lifespan but also to enhance the overall quality of life for individuals afflicted with LSDs. This review provides insights into lysosomal dysfunction from a molecular perspective, particularly in the context of human diseases, and highlights recent advancements and breakthroughs in this field.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with limited treatment options. ALS pathogenesis involves intricate processes within motor neurons (MNs), characterized by dysregulated Ca2+ influx and buffering in early ALS-affected MNs. This study proposes the modulation of ryanodine receptors (RyRs), key mediators of intracellular Ca2+, as a therapeutic target. A novel class of novel FKBP12 ligands that show activity as cytosolic calcium modulators through stabilizing RyR channel activity, were tested in the SOD1-G93A mouse model of ALS. Different outcomes were used to assess treatment efficacy including electrophysiology, histopathology, neuromuscular function, and survival. Among the novel FKBP12 ligands, MP-010 was chosen for its central nervous system availability. Chronic administration of MP-010 to SOD1-G93A mice produced a dose-dependent preservation of motor nerve conduction, with the 61 mg/kg dose significantly delaying the onset of motor impairment. This was accompanied by improved motor coordination, increased innervated endplates, and significant preservation of MNs in the spinal cord of treated mice. Notably, MP-010 treatment significantly extended lifespan by an average of 10 days compared to vehicle. In conclusion, FKBP12 ligands, particularly MP-010, exhibit promising neuroprotective effects in ALS, highlighting their potential as novel therapeutic agents. Further investigations into the molecular mechanisms and clinical translatability of these compounds are needed for their application in ALS treatment.

This project involves creating a real-time geo-referenced measurement platform for kindergartens, utilizing IoT technology and the WebSockets protocol to enhance environmental information in socially significant areas. The platform displays air quality data on a map and provides Twitter alerts for local pollution events. Tests in two kindergartens demonstrated effective data capture and transmission to a central server, achieving a 92% success rate at distances exceeding 800 meters. The platform also distinguished environmental differences between the kindergartens based on location, proving its utility in supporting parents and guardians in selecting kindergartens for their children.

In the original version of this article, the authors wrongly assembled panels for some of the Western Blots presented. Additionally, the stated number of experiments conducted requires correction as per below. In Figure 1d, the chosen blot depicting p62 expression does not originate from the same samples illustrating Beclin-1, LC3, and the loading control. The correct representative images for the Western Blot experiments are shown below. The legend for Figure 1 is changed from: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of 10 independent experiments. […]” To: “[…] Results are expressed as mean ± SEM, and with blots are representative of three independent experiments. […]” In Figure 3, the chosen blots depicting the loading controls do not originate from the same samples illustrating eIFα and Ser51P-eIF2α in panel a, CHOP in panel b, and IRE1α in panel c. The corrected panels are shown below. The legend for Figure 3 is changed from: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of five independent experiments. […]” To: “[…] Results are expressed as mean ± SEM, and with blots are representative of six to nine independent experiments. […]” In Figure 5c, duplications have occurred due to mistakes during figure compilation. The correct representative images for the Western Blot experiments are shown below. The legend for Figure 5 is changed from: “[…] Results are expressed as mean ± standard error of the mean, and with the blots and images are representative of four independent experiments. […]” To: “[…] Results are expressed as mean ± standard error of the mean, and with the blots and images are representative of three to five independent experiments. […]” In Figure 7d, the chosen blots depicting JNK1/2 expression for samples treated with SP600125 and Sorafenib+SP600125 at 12 and 24 h have been mistakenly inverted. The correct representative images for the Western Blot experiments are shown below. The legend for Figure 7 is changed from: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of four independent experiments. […]” To: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of three independent experiments. […]” In Figure 8c, the authors omitted the indication of separation between non-consecutively loaded samples. The correct representative images for the Western Blot experiments are shown below. The legend for Figure 8 is changed from: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of eight independent experiments. […]” To: “[…] Results are expressed as mean ± standard error of the mean, and with the blots are representative of three to six independent experiments. […]” This correction doesn't change the results and conclusions. The authors apologize for any confusion these errors may have caused.

Background: Vitamin D receptor (VDR) is a nuclear hormone receptor widely expressed in the substantia nigra. Its association with an increased risk of Parkinson's disease (PD) is based on vitamin D deficiency and/or different polymorphisms in its gene receptor. This fact has been demonstrated by several case-control studies. Materials and Methods: Consequently, we investigated the association between VDR ApaI, BsmI, FokI, and TaqI gene polymorphisms and PD in a Spanish cohort that included 54 cases and 17 healthy controls. The detection of single nucleotide polymorphisms (SNPs) was performed using a polymerase chain reaction-restriction fragment length polymorphism. Results: Our data indicate that the SNPs were not associated with the age of onset of PD, nor with the occurrence of motor symptoms. However, only BsmI polymorphism was significantly associated with PD in this Spanish cohort. In fact, BsmI genotype was five times higher among PD patients than among controls, and the A allele was considered as a genetic risk for PD. Additionally, the combination of FokI and BsmI polymorphisms was significantly associated with PD and could represent a risk factor. Conclusion: We conclude that ApaI, TaqI, and FokI polymorphisms were not associated with PD, but BsmI could be a risk factor for PD in this randomized population.

Background and Purpose: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with limited treatment options. ALS pathogenesis involves intricate processes within motor neurons (MNs), characterized by dysregulated Ca2+ influx and buffering in early ALS-affected MNs. This study proposes the modulation of ryanodine receptors (RyRs), key mediators of intracellular Ca2+, as a therapeutic target. Experimental Approach: A novel class of novel FKBP12 ligands that show activity as cytosolic calcium modulators through stabilizing RyR channel activity, were tested in the SOD1G93A mouse model of ALS. Different outcomes were used to assess treatment efficacy including electrophysiology, histopathology, neuromuscular function, and survival. Key Results: Among the novel FKBP12 ligands, MP-010 was chosen for its central nervous system availability. Chronic administration of MP-010 to SOD1G93A mice produced a dose-dependent preservation of motor nerve conduction, with the 61 mg/kg dose significantly delaying the onset of motor impairment. This was accompanied by improved motor coordination, increased innervated endplates, and significant preservation of MNs in the spinal cord of treated mice. Notably, MP-010 treatment significantly extended lifespan by an average of 10 days compared to vehicle. Conclusions and Implications: FKBP12 ligands, particularly MP-010, exhibit promising neuroprotective effects in ALS, highlighting their potential as novel therapeutic agents. Further investigations into the molecular mechanisms and clinical translatability of these compounds are needed for their application in ALS treatment.

The soil nematode worm Caenorhabditis elegans is a simple and well-established model for the study of many biological processes. Heat shock and thermotolerance assays have been developed for this nematode, and have been used to decipher the molecular relationships between thermal stress and aging, among others. Nevertheless, a systematic and methodological comparison of the different approaches and tools utilized is lacking in the literature. Here, we aim to provide a comprehensive summary of the most commonly used strategies for carrying out heat shock and thermotolerance assays that have been reported, highlighting specific readouts and scientific questions that can be addressed. Furthermore, we offer examples of thermotolerance assays performed with wild type nematodes, that can serve as a gauge of the animal survival under diverse conditions of stress.

Cardiolipin (CL) is a mitochondria-exclusive phospholipid synthesized in the inner mitochondrial membrane. CL plays a key role in mitochondrial membranes, impacting a plethora of functions this organelle performs. Consequently, it is conceivable that abnormalities in the CL content, composition, and level of oxidation may negatively impact mitochondrial function and dynamics, with important implications in a variety of diseases. This review concentrates on papers published in recent years, combined with basic and underexplored research in CL. We capture new findings on its biological functions in the mitochondria, as well as its association with neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Lastly, we explore the potential applications of CL as a biomarker and pharmacological target to mitigate mitochondrial dysfunction.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of the CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. This expansion leads to a polyglutamine (polyQ) tract at the N-terminal end of HTT, which reduces the solubility of the protein and promotes its accumulation. Inefficient clearance of mutant HTT (mHTT) by the proteasome or autophagy–lysosomal system leads to accumulation of oligomers and toxic protein aggregates in neurons, resulting in impaired proteolytic systems, transcriptional dysregulation, impaired axonal transport, mitochondrial dysfunction and cellular energy imbalance. Growing evidence suggests that the accumulation of mHTT aggregates and autophagic and/or lysosomal dysfunction are the major pathogenic mechanisms underlying HD. In this context, enhancing autophagy may be an effective therapeutic strategy to remove protein aggregates and improve cell function. Transcription factor EB (TFEB), a master transcriptional regulator of autophagy, controls the expression of genes critical for autophagosome formation, lysosomal biogenesis, lysosomal function and autophagic flux. Consequently, the induction of TFEB activity to promote intracellular clearance may be a therapeutic strategy for HD. However, while some studies have shown that overexpression of TFEB facilitates the clearance of mHTT aggregates and ameliorates the disease phenotype, others indicate such overexpression may lead to mHTT co-aggregation and worsen disease progression. Further studies are necessary to confirm whether TFEB modulation could be an effective therapeutic strategy against mHTT-mediated toxicity in different disease models.

The etiology of various neurodegenerative disorders that mainly affect the central nervous system including (but not limited to) Alzheimer's disease, Parkinson's disease and Huntington's disease has classically been attributed to neuronal defects that culminate with the loss of specific neuronal populations. However, accumulating evidence suggests that numerous immune effector cells and the products thereof (including cytokines and other soluble mediators) have a major impact on the pathogenesis and/or severity of these and other neurodegenerative syndromes. These observations not only add to our understanding of neurodegenerative conditions but also imply that (at least in some cases) therapeutic strategies targeting immune cells or their products may mediate clinically relevant neuroprotective effects. Here, we critically discuss immunological mechanisms of central neurodegeneration and propose potential strategies to correct neurodegeneration-associated immunological dysfunction with therapeutic purposes.

The cultivation of the mussel, Mytilus galloprovincialis, in the Galician Rı́as (NW Spain) requires approximately 4600 tonnes of mussel seed every year. Traditionally, this seed is obtained from two different sources: intertidal rocky shore and collector ropes. In this study, the biological performance (growth rate, mortality and parasitization) of mussels from both sources of seed was compared in a cultivation area of the Rı́a de Arousa. The results from this experiment indicate there is no difference in biological performance between the two types of mussels seed under standard cultivation conditions. Nevertheless, the use of mussel seed from collector ropes is recommended due to their significantly larger size at harvest time. The main deficiency of this type of seed, its high mortality from seeding to thinning out, could be offset by improving the procedure of tying the mussels to culture ropes.

Aldohexose derivatives possessing a triphenylphosphoranylideneamino substituent at the primary (C-6) position exhibit an increased reactivity towards the aza-Wittig condensation with isothiocyanates as compared with glycosyl phosphinimines. The reaction is particularly fast and efficient in the case of glycosyl isothiocyanates and has been exploited in the preparation of (1→6)-linked pseudooligosaccharides incorporating carbodiimide bridges. These compounds are excellent starting materials for the preparation of sugar ureas, thioureas and guanidines by reaction with the appropriate nucleophile.

Abstract The reaction of aldose oligosaccharides with aqueous ammonium hydrogencarbonate was revised and optimized for gram scale preparation of glycosylamines, using lactose, cellobiose, maltose, and melibiose as model compounds. The unprotected glycosylamines 1b-4b were transformed into the corresponding hepta-O-acetyl hydrochlorides 1e-4e via diethyl N-glycosylaminomethylenemalonates (1c-4c and 1d-4d). Reaction of le-3e with thiophosgene in a three-phased system led to the target hepta-O-acetylglycosyl isothiocyanates lf-3f in excellent yields. Under the same conditions, the melibiosyl derivative 4e underwent partial hydrolysis to give a mixture of melibiosyl isothiocyanate 4f and the reducing sugar derivative 4h. Treatment of 1f-4f with ammonia resulted in the quantitative formation of N-glycosylthioureas (1g-4g).

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEnantiopure 2-Thioxotetrahydro-1,3-O,N-heterocycles from Carbohydrates. 3. Enantiopure C-4 Chiral Oxazine- and Oxazolidine-2-thiones from 3-Deoxy-3-isothiocyanato SugarsJose M. Garcia Fernandez, Carmen Ortiz Mellet, Jose L. Jimenez Blanco, and Jose FuentesCite this: J. Org. Chem. 1994, 59, 19, 5565–5572Publication Date (Print):September 1, 1994Publication History Published online1 May 2002Published inissue 1 September 1994https://pubs.acs.org/doi/10.1021/jo00098a014https://doi.org/10.1021/jo00098a014research-articleACS PublicationsRequest reuse permissionsArticle Views581Altmetric-Citations30LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

The preparation of [(2S,3S,4R)-3,4-dihydroxypyrrolidin-2-yl]furan derivatives in a stereoselective route starting from d-glucose and ethyl acetoacetate is presented. Ethyl ester (6), N,N-diethylamide (7) and N-isopropylamide (8) have been tested towards 25 glycosidases. Ester (6) is a selective inhibitor of β-galactosidases. The new compounds represent a new type of imino-C-nucleoside analogues.