In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson's disease and other related ...disorders, such as Dementia with Lewy Bodies and Alzheimer's disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated ...alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown.
In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T).
These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, ...although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
In the context of time domain astronomy, we present an offline detection search of gamma-ray transients using a wild binary segmentation analysis called F-WBSB targeting both short and long ...gamma-ray bursts (GRBs) and covering the soft and hard gamma-ray bands. We use NASA Fermi/GBM archival data as a training and testing data set. This paper describes the analysis applied to the 12 NaI detectors of the Fermi/GBM instrument. This includes background removal, change-point detection that brackets the peaks of gamma-ray flares, the evaluation of significance for each individual GBM detector, and the combination of the results among the detectors. We also explain the calibration of the ∼ 10 parameters present in the method using one week of archival data. Finally, we present our detection performance result for 60 d of a blind search analysis with F-WBSB by comparing to both the onboard and offline GBM search as well as external events found by others surveys such as Swift-BAT. We detect 42/44 onboard GBM events but also other gamma-ray flares at a rate of 1 per hour in the 4–50 keV band. Our results show that F-WBSB is capable of recovering gamma-ray flares, including the detection of soft X-ray long transients. FWBSB offers an independent identification of GRBs in combination with methods for determining spectral and temporal properties of the transient as well as localization. This is particularly useful for increasing the GRB rate and that will help the joint detection with gravitational-wave events.
Abstract In Parkinson's disease (PD) depletion of dopamine in the nigro-striatal pathway is a main pathological hallmark that requires continuous and focal restoration. Current predominant treatment ...with intermittent oral administration of its precursor, Levodopa ( l -dopa), remains the gold standard but pharmacological drawbacks trigger motor fluctuations and dyskinesia. Continuous intracerebroventricular (i.c.v.) administration of dopamine previously failed as a therapy because of an inability to resolve the accelerated dopamine oxidation and tachyphylaxia. We aim to overcome prior challenges by demonstrating treatment feasibility and efficacy of continuous i.c.v. of dopamine close to the striatum. Dopamine prepared either anaerobically (A-dopamine) or aerobically (O-dopamine) in the presence or absence of a conservator (sodium metabisulfite, SMBS) was assessed upon acute MPTP and chronic 6-OHDA lesioning and compared to peripheral l -dopa treatment. A-dopamine restored motor function and induced a dose dependent increase of nigro-striatal tyrosine hydroxylase positive neurons in mice after 7 days of MPTP insult that was not evident with either O-dopamine or l -dopa. In the 6-OHDA rat model, continuous circadian i.c.v. injection of A-dopamine over 30 days also improved motor activity without occurrence of tachyphylaxia. This safety profile was highly favorable as A-dopamine did not induce dyskinesia or behavioral sensitization as observed with peripheral l -dopa treatment. Indicative of a new therapeutic strategy for patients suffering from l -dopa related complications with dyskinesia, continuous i.c.v. of A-dopamine has greater efficacy in mediating motor impairment over a large therapeutic index without inducing dyskinesia and tachyphylaxia.
In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). ...In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.
Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet beta cells. Despite many efforts focused on identifying new therapies able to counteract this ...autoimmune attack and/or stimulate beta cells regeneration, TD1M remains without effective clinical treatments providing no clear advantages over the conventional treatment with insulin. We previously postulated that both the inflammatory and immune responses and beta cell survival/regeneration must be simultaneously targeted to blunt the progression of disease. Umbilical cord-derived mesenchymal stromal cells (UC-MSC) exhibit anti-inflammatory, trophic, immunomodulatory and regenerative properties and have shown some beneficial yet controversial effects in clinical trials for T1DM. In order to clarify conflicting results, we herein dissected the cellular and molecular events derived from UC-MSC intraperitoneal administration (i.p.) in the RIP-B7.1 mouse model of experimental autoimmune diabetes. Intraperitoneal (i.p.) transplantation of heterologous mouse UC-MSC delayed the onset of diabetes in RIP-B7.1 mice. Importantly, UC-MSC i. p. transplantation led to a strong peritoneal recruitment of myeloid-derived suppressor cells (MDSC) followed by multiple T-, B- and myeloid cells immunosuppressive responses in peritoneal fluid cells, spleen, pancreatic lymph nodes and the pancreas, which displayed significantly reduced insulitis and pancreatic infiltration of T and B Cells and pro-inflammatory macrophages. Altogether, these results suggest that UC-MSC i. p. transplantation can block or delay the development of hyperglycemia through suppression of inflammation and the immune attack.
Human pluripotent stem cells retain the extraordinary capacity to differentiate into pancreatic beta cells. For this particular lineage, more effort is still required to stress the importance of ...developing an efficient, reproducible, easy, and cost-effective differentiation protocol to obtain more mature, homogeneous, and functional insulin-secreting cells. In addition, microRNAs (miRNAs) have emerged as a class of small non-coding RNAs that regulate many cellular processes, including pancreatic differentiation. Some miRNAs are known to be preferentially expressed in islets. Of note, miR-375 and miR-7 are two of the most abundant pancreatic miRNAs, and they are necessary for proper pancreatic islet development. Here we provide new insight into specific miRNAs involved in pancreatic differentiation. We found that miR-7 is differentially expressed during the differentiation of human embryonic stem cells (hESCs) into a beta cell-like phenotype and that its modulation plays an important role in generating mature pancreatic beta cells. This strategy may be exploited to optimize the potential for in vitro differentiation of hESCs into insulin-producing beta-like cells for use in preclinical studies and future clinical applications as well as the prospective uses of miRNAs to improve this process.
Context. Ultra-high-energy cosmic rays (UHECRs) have attracted a lot of attention in astroparticle physics and high-energy astrophysics, due to their challengingly high energies, and to their ability ...to constrain the physical processes and astrophysical parameters in the most energetic sources of the universe. Despite their very large acceptance, current detectors have failed to detect significant anisotropies in their arrival directions, which had been expected to lead to the long-sought identification of their sources. Some indications about the composition of the UHECRs, which may become heavier at the highest energies, have even called into question the possibility that such a goal could be achieved in the foreseeable future. Aims. We investigate the potential value of a new-generation detector, with an exposure increased by one order of magnitude, to overcome the current situation and make notable progress in detecting anisotropies and thus in the study of UHECRs. We take as an example the expected performances of the JEM-EUSO detector, assuming a uniform full-sky coverage with a total exposure of 300 000 km2 sr yr. Methods. We simulated realistic UHECR sky maps for a wide range of possible astrophysical scenarios allowed by the current constraints, taking the energy losses and photo-dissociation of the UHE protons and nuclei into account, as well as their deflections by intervening magnetic fields. These sky maps, built for both the expected statistics of JEM-EUSO and the current Pierre Auger Observatory statistics, as a reference, were analysed from the point of view of their intrinsic anisotropies, using the two-point correlation function. A statistical study of the resulting anisotropies was performed for each astrophysical scenario, varying the UHECR source composition and spectrum and the source density and exploring a set of five hundred independent realizations for each choice of a parameter set. Results. We find that significant anisotropies are expected to be detected by a next-generation UHECR detector, for essentially all the astrophysical scenarios studied, and give precise, quantitative meaning to this statement. Conclusions. Our results show that a gain of one order of magnitude in the total exposure of UHECR detectors would make a significant difference compared to the existing experiments, and would allow considerable progress in the study of these mysterious particles and their sources.
BACKGROUND: Features of both apoptosis and necrosis have been reported in ejaculated human spermatozoa. This study examines the relative contribution of these two modes of cell death to the demise of ...these terminally differentiated cells. METHODS: Sperm fractions were prepared from aliquots of semen samples from young normozoospermic donors by simple washing from seminal plasma, by discontinuous density gradient centrifugation or by swim‐up technique. They were subsequently incubated in vitro at 37°C for 24 h. Sperm motility, viability, and the presence of two apoptotic markers, phosphatidylserine externalization (annexin‐V binding) and DNA fragmentation (TUNEL), were examined before incubation and again after 4 and 24 h of incubation. RESULTS: The swim‐up technique was the most efficient in terms of the recovery of viable, motile and non‐apoptotic spermatozoa, followed by density gradient centrifugation and finally simple washing. No changes in the parameters tested were observed after 4 h of incubation, but a significant decrease in sperm motility and viability was detected after 24 h irrespective of the sperm preparation technique employed. However, these changes were not accompanied by any increase in the incidence of spermatozoa showing markers of an active apoptotic process. CONCLUSIONS: Healthy human ejaculated spermatozoa appear incapable of initiating apoptosis, at least under in vitro conditions.
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