Paired helical filaments from Alzheimer disease brain induce intracellular accumulation of Tau protein in aggresomes

Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology.     

Caprylic Triglyceride as a Novel Therapeutic Approach to Effectively Improve the Performance and Attenuate the Symptoms Due to the Motor Neuron Loss in ALS Disease

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and finally death. ALS patients suffer from asthenia and their progressive weakness negatively impacts quality of life, limiting their daily activities. They have impaired energy balance linked to lower activity of mitochondrial electron transport chain enzymes in ALS spinal cord, suggesting that improving mitochondrial function may present a therapeutic approach for ALS. When fed a ketogenic diet, the G93A ALS mouse shows a significant increase in serum ketones as well as a significantly slower progression of weakness and lower mortality rate. In this study, we treated SOD1-G93A mice with caprylic triglyceride, a medium chain triglyceride that is metabolized into ketone bodies and can serve as an alternate energy substrate for neuronal metabolism. Treatment with caprylic triglyceride attenuated progression of weakness and protected spinal cord motor neuron loss in SOD1-G93A transgenic animals, significantly improving their performance even though there was no significant benefit regarding the survival of the ALS transgenic animals. We found that caprylic triglyceride significantly promoted the mitochondrial oxygen consumption rate in vivo. Our results demonstrated that caprylic triglyceride alleviates ALS-type motor impairment through restoration of energy metabolism in SOD1-G93A ALS mice, especially during the overt stage of the disease. These data indicate the feasibility of using caprylic acid as an easily administered treatment with a high impact on the quality of life of ALS patients.     

FRUIT,a Scar-Free System for Targeted Chromosomal Mutagenesis,Epitope Tagging,and Promoter Replacement in Escherichia coli and Salmonella enterica

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.     

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.     

Modification of Emission Properties of ZnO Layers due to Plasmonic Near-Field Coupling to Ag Nanoislands

A simple fabrication method of silver (Ag) nanoislands on ZnO films is presented. Continuous wave and time-resolved photoluminescence and transmission are employed to investigate modifications of visible and UV emissions of ZnO brought about by coupling to localized surface plasmons residing on Ag nanoislands. The size of the nanoislands, determining their absorption and scattering efficiencies, is found to be an important factor governing plasmonic modification of optical response of ZnO films. The presence of the Ag nanoislands of appropriate dimensions causes a strong (threefold) increase in emission intensity and up to 1.5 times faster recombination. The experimental results are successfully described by model calculations within the Mie theory.     

Magnesium permeation through mechanosensitive channels： single-current measurements

Compelling evidence shows that intracellular free magnesium [Mg^2＋]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg^2＋ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive （MS） cationic channels activated by stretch are permeable for Mg^2＋ ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl2 when Mg^2＋ was the only available carrier of inward currents. At 2 mM Mg^2＋, inward mechanogated currents representing Mg^2＋ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg^2＋ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg^2＋ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg^2＋, mean conductance values for inward currents carried by Mg^2＋ were rather similar, being equal to 6.8 ± 0.5 and 6.4 ± 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg^2＋ permeation through membrane channels activated by stretch. Our single-current measurements document Mg^2＋ influx through MS channels in the plasma membrane of leukaemia cells.     

Toxoplasma gondii seroprevalence in goats,cats and humans in Russia

Toxoplasmosis, a most common zoonosis, is caused by the protozoan parasite Toxoplasma gondii. However, there is little epidemiological information on T. gondii infections in humans and livestock animals in Russia. Therefore, in this study, the seroprevalence of T. gondii in goats in Russia was investigated. A total of 216 goats from 32 farms were investigated and 95 of them were seropositive for T. gondii. The difference in seroprevalence between the examined regions was not statistically significant. We next collected serum samples from 99 cats and 181 humans in Kazan city, the state capital of the Republic of Tatarstan, Russia, and examined their T. gondii seroprevalences. Thirty-nine of the 99 cat samples and 56 of the 181 human samples showed seropositivity. Logistical regression analysis revealed that the cat breeding history of the human subjects, but not their sex or age is a significant risk factor for T. gondii seropositivity. These findings suggest that the natural environment in Russia may be widely polluted with T. gondii oocysts shed by cats, and ingestion of these oocysts provides a major route for human infection with this parasite.     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.