Implication of the NLRP3 Inflammasome in Bovine Age-Related Sarcopenia

Sarcopenia is defined as the age-related loss of skeletal muscle mass, quality, and strength. The pathophysiological mechanisms underlying sarcopenia are still not completely understood. The aim of this work was to evaluate, for the first time, the expression of NLRP3 inflammasome in bovine skeletal muscle in order to investigate the hypothesis that inflammasome activation may trigger and sustain a pro-inflammatory environment leading to sarcopenia. Samples of skeletal muscle were collected from 60 cattle belonging to three age-based groups. Morphologic, immunohistochemical and molecular analysis were performed to assess the presence of age-related pathologic changes and chronic inflammation, the expression of NLRP3 inflammasome and to determine the levels of interleukin-1β, interleukin-18 and tumor necrosis factor alpha in muscle tissue. Our results revealed the presence of morphologic sarcopenia hallmark, chronic lymphocytic inflammation and a type II fibers-selective NLRP3 expression associated to a significant decreased number of immunolabeled-fibers in aged animals. Moreover, we found a statistically significant age-related increase of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 suggesting the activation of NLRP3 inflammasome. Taken together, our data suggest that NLRP3 inflammasome components may be normally expressed in skeletal muscle, but its priming and activation during aging may contribute to enhance a pro-inflammatory environment altering normal muscular anabolism and metabolism.     

Novel KCND3 Variant Underlying Nonprogressive Congenital Ataxia or SCA19/22 Disrupt KV4.3 Protein Expression and K+ Currents with Variable Effects on Channel Properties

KCND3 encodes the voltage-gated potassium channel K_V4.3 that is highly expressed in the cerebellum, where it regulates dendritic excitability and calcium influx. Loss-of-function K_V4.3 mutations have been associated with dominant spinocerebellar ataxia (SCA19/22). By targeted NGS sequencing, we identified two novel KCND3 missense variants of the K_V4.3 channel: p.S347W identified in a patient with adult-onset pure cerebellar syndrome and p.W359G detected in a child with congenital nonprogressive ataxia. Neuroimaging showed mild cerebellar atrophy in both patients. We performed a two-electrode voltage-clamp recording of K_V4.3 currents in Xenopus oocytes: both the p.G345V (previously reported in a SCA19/22 family) and p.S347W mutants exhibited reduced peak currents by 50%, while no K+ current was detectable for the p.W359G mutant. We assessed the effect of the mutations on channel gating by measuring steady-state voltage-dependent activation and inactivation properties: no significant alterations were detected in p.G345V and p.S347W disease-associated variants, compared to controls. K_V4.3 expression studies in HEK293T cells showed 53% (p.G345V), 45% (p.S347W) and 75% (p.W359G) reductions in mutant protein levels compared with the wildtype. The present study broadens the spectrum of the known phenotypes and identifies additional variants for KCND3-related disorders, outlining the importance of SCA gene screening in early-onset and congenital ataxia.     

The Effects Of Humic Substances On Pinus Callus Are Reversed By 2,4-Dichlorophenoxyacetic Acid

The reversal of humic matter-induced inhibition of callus growth and metabolism by 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in Pinus laricio. Two forest humic fractions (relative molecular mass (M_r) > 3500), derived from soil under Fagus sylvatica (Fs) and Abies alba (Aa) plantation, were used. Pinus laricio callus was grown for a subculture period (4 weeks) on Basal Murashige and Skoog (MS) medium plus forest humic matters (Fs or Aa), at a concentration of 1 mg C/l, and then was transferred, for an additional four weeks, to a MS medium culture without humic matter, but with different hormones: indole-3-acetic acid (IAA, 2 mg/1) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/1) and/or 6-benzylaminopurine (BAP, 0.25 mg/1). Growth of calluse, glucose, fructose, and sucrose contents, and activities of soluble and bound invertases, glucokinase, phosphoglucose isomerase, aldolase, and pyruvate kinase were monitored. The results show a negative effect of humic fractions on callus growth, due to decreased utilization of glucose and fructose, and decreased activities of glycolytic enzymes. The effects are reversible. Substitution of humic fractions with 2,4-D+BAP or 2,4-D is followed by an increase of glycolytic enzyme activities and, consequently, by the utilization of glucose and fructose that induces a restart of growth. In contrast, the inhibitory effects of humic fractions persist when they are substituted with BAP alone, indicating that only the auxin 2,4-D is capable of reversing the negative effects. A possible competitive action on the auxin-binding site between 2,4-D and the chemical structures in the forest humic fractions is suggested.     

Temperature during conservation in laboratory silos affects fermentation profile and aerobic stability of corn silage treated with Lactobacillus buchneri,Lactobacillus hilgardii,and their combination

The environment temperature and its effect on the temperature of silage is very important for the fermentation and subsequent quality of a silage. Obligate heterofermentative lactic acid bacteria (LAB) inocula, because of their ability to inhibit yeasts, have been developed to prevent the aerobic deterioration of silages. The temperature during silage conservation may also play an important role in the fermentation profile of silages. This study has evaluated the effect of temperature, during the conservation of whole crop corn silage, untreated or treated with different LAB inocula, on the fermentation profile and on the aerobic stability of the silage. Corn was harvested at 42% dry matter and either not treated (control) or treated with Lactobacillus buchneri NCIMB 40788 (LB) at 300,000 cfu/g fresh matter (FM); Lactobacillus hilgardii CNCM I-4785 at 150,000 cfu/g FM (LH₁₅₀); L. hilgardii CNCM I-4785 at 300,000 cfu/g FM (LH₃₀₀); or LB+LH at 150,000 cfu/g FM each. In an attempt to experimentally simulate temperature fluctuations in the mass or at the periphery of a silage bunker, corn was conserved in laboratory silos at a constant temperature (20 ± 1°C; MASS) or at lower and variable outdoor temperatures (PERIPH; ranging from 0.5 to 19°C), and the silos were opened after 15, 30, and 100 d of conservation. Lactic acid, acetic acid, and ethanol contents increased in all the silages over the conservation period. The lactic acid content was higher (+10%) in the silages kept at a constant temperature than those conserved at the lower and variable outdoor temperatures. The acetic acid was higher in the treated silages than in the control ones conserved at a constant temperature for 100 d. Moreover, 1,2-propanediol was only detected in the treated silages after at least 30 d at a constant temperature, whereas only traces were detected in the LB+LH treatment for the other temperature conditions. The yeast count decreased during conservation at a slower rate in PERIPH than in MASS and on average reached 2.96 and 4.71 log cfu/g for MASS and PERIPH, respectively, after 100 d of conservation. The highest aerobic stability values were observed for LH₃₀₀ (191 h) in the MASS silage after 100 d of conservation, whereas the highest aerobic stability was observed in LB+LH (150 h) in the PERIPH silages. After 7 d of air exposure, a pH higher than 4.5 and a higher yeast than 8.0 log cfu/g were detected in all the silages opened after 15 and 30 d of conservation. A pH value close to that of silo opening was detected in the LB, LH₁₅₀, and LH₃₀₀ silages conserved under MASS conditions after 100 d, whereas LB+LH was the most effective under PERIPH conditions. The temperature and its fluctuation during conservation of silage in laboratory silos influenced the fermentation, which in turn had an effect on the quality of silage and on the extent of the effect of LAB inocula.     

Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH₂), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.     

A covert attention P300-based brain-computer interface: Geospell

The Farwell and Donchin P300 speller interface is one of the most widely used brain-computer interface (BCI) paradigms for writing text. Recent studies have shown that the recognition accuracy of the P300 speller decreases significantly when eye movement is impaired. This report introduces the GeoSpell interface (Geometric Speller), which implements a stimulation framework for a P300-based BCI that has been optimised for operation in covert visual attention. We compared the Geospell with the P300 speller interface under overt attention conditions with regard to effectiveness, efficiency and user satisfaction. Ten healthy subjects participated in the study. The performance of the GeoSpell interface in covert attention was comparable with that of the P300 speller in overt attention. As expected, the effectiveness of the spelling decreased with the new interface in covert attention. The NASA task load index (TLX) for workload assessment did not differ significantly between the two modalities. PRACTITIONER SUMMARY: This study introduces and evaluates a gaze-independent, P300-based brain-computer interface, the efficacy and user satisfaction of which were comparable with those off the classical P300 speller. Despite a decrease in effectiveness due to the use of covert attention, the performance of the GeoSpell far exceeded the threshold of accuracy with regard to effective spelling.     

Protein Expression Changes in Maize Roots in Response to Humic Substances

Humic substances are known to affect plant metabolism at different levels. We characterized humic substances extracted from earthworm feces by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy and used them to treat corn, Zea mays L., seedlings to investigate changes in patterns of root protein expression. After root plasma membrane extraction and purification, proteins were separated by two-dimensional gel electrophoresis, and differential spot intensities were evaluated by image analysis. Finally, 42 differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The majority of them were downregulated by the treatment with humic substances. The proteins identified included malate dehydrogenase, ATPases, cytoskeleton proteins, and different enzymes belonging to the glycolytic/gluconeogenic pathways and sucrose metabolism. The identification of factors involved in plant responses to humic substances may improve our understanding of plant-soil cross-talk, and enable a better management of soil resources.     

Low-molecular-weight organic acids and hormone-like activity of dissolved organic matter in two forest soils in N Italy

Concentrations of aliphatic acids, phenolic acid, and inorganic nutrients, as well as hormone-like activity, were determined in soil dissolved organic matter (DOM) from two forest sites in N Italy showing differing degrees of silver fir regeneration. In the site where silver fir recruitment was abundant, humification processes prevailed, and the soil DOM had a high content in aliphatic and phenolic acids. This enhanced the hormone-like activity in the soil, which could in turn promote growth of silver fir seedlings. In the site with poor fir recruitment, the soil DOM underwent rapid mineralization and was richer in inorganic nutrients, but had lower concentrations of aliphatic and phenolic acids, and lower hormone-like activity.