Biocomposites Based on Polyamide 11/Diatoms with Different Sized Frustules.

Amorphous diatomite was used as a filler for a thermoplastic polymer of polyamide 11 obtained from natural sources. The diatomite particles of different sizes were previously fractionated by sedimentation to obtain powders with varying particle size distribution, including powders with or without frustule particles, crushed, uncrushed or agglomerated. Biocomposites containing 2.5, 5, 10 and 20% filler were tested for their mechanical properties, including tensile strength, flexural strength and impact strength. In addition, a particle size analysis (by Dynamic Light Scattering, DLS) was performed and the dispersion of the filler in the polymer matrix (Scanning Electron Microscopy, SEM), thermal parameters (Differential Scanning Calorimetry, DSC, and Dynamic Mechanical Analysis, DMA) were determined. Testing showed that biocomposites modified with diatomaceous earth have a higher mechanical strength than the reference system, especially with larger amounts of the filler (10 and 20%), e.g., the tensile strength of pure PA11 is about 46 MPa, while 20OB and 20OF 47.5 and 47 MPa, respectively, while an increase in max. flexural strength and flexural modulus is also observed compared to pure PA11 by a maximum of 63 and 54%, respectively Diatomaceous earth can be obtained in various ways-it is commercially available or it is possible to breed diatoms in laboratory conditions, while the use of commercially available diatomite, which contains diatoms of different sizes, eliminates the possibility of controlling mechanical parameters by filling biocomposites with a filler with the desired particle size distribution, and diatom breeding is not possible on an industrial scale. Our proposed biocomposite based on fractionated diatomaceous earth using a sedimentation process addresses the current need to produce biocomposite materials from natural sources, and moreover, the nature of the process, due to its simplicity, can be successfully used on an industrial scale.

The serotonin 2B receptor is required in neonatal microglia to limit neuroinflammation and sickness behavior in adulthood.

Severe peripheral infections induce an adaptive sickness behavior and an innate immune reaction in various organs including the brain. On the long term, persistent alteration of microglia, the brain innate immune cells, is associated with an increased risk of psychiatric disorders. It is thus critical to identify genes and mechanisms controlling the intensity and duration of the neuroinflammation induced by peripheral immune challenges. We tested the hypothesis that the 5-HT2B receptor, the main serotonin receptor expressed by microglia, might represent a valuable candidate. First, we observed that Htr2b-/- mice, knock-out for the 5-HT2B receptor gene, developed, when exposed to a peripheral lipopolysaccharide (LPS) challenge, a stronger weight loss compared to wild-type mice; in addition, comparison of inflammatory markers in brain, 4 and 24 hr after LPS injection, showed that Htr2b deficiency leads to a prolonged neuroinflammation. Second, to assess the specific contribution of the microglial 5-HT2B receptor, we investigated the response to LPS of conditional knock-out mice invalidated for Htr2b in microglia only. We found that deletion of Htr2b in microglia since birth is sufficient to cause enhanced weight loss and increased neuroinflammatory response upon LPS injection at adult stage. In contrast, mice deleted for microglial Htr2b in adulthood responded normally to LPS, revealing a neonatal developmental effect. These results highlight the role of microglia in the response to a peripheral immune challenge and suggest the existence of a developmental, neonatal period, during which instruction of microglia through 5-HT2B receptors is necessary to prevent microglia overreactivity in adulthood.

m-AAA Complexes Are Not Crucial for the Survival of Arabidopsis Under Optimal Growth Conditions Despite Their Importance for Mitochondrial Translation.

For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.

AtOMA1 Affects the OXPHOS System and Plant Growth in Contrast to Other Newly Identified ATP-Independent Proteases in Arabidopsis Mitochondria.

Compared with yeast, our knowledge on members of the ATP-independent plant mitochondrial proteolytic machinery is rather poor. In the present study, using confocal microscopy and immunoblotting, we proved that homologs of yeast Oma1, Atp23, Imp1, Imp2, and Oct1 proteases are localized in Arabidopsis mitochondria. We characterized these components of the ATP-independent proteolytic system as well as the earlier identified protease, AtICP55, with an emphasis on their significance in plant growth and functionality in the OXPHOS system. A functional complementation assay demonstrated that out of all the analyzed proteases, only AtOMA1 and AtICP55 could substitute for a lack of their yeast counterparts. We did not observe any significant developmental or morphological changes in plants lacking the studied proteases, either under optimal growth conditions or after exposure to stress, with the only exception being retarded root growth in oma1-1, thus implying that the absence of a single mitochondrial ATP-independent protease is not critical for Arabidopsis growth and development. We did not find any evidence indicating a clear functional complementation of the missing protease by any other protease at the transcript or protein level. Studies on the impact of the analyzed proteases on mitochondrial bioenergetic function revealed that out of all the studied mutants, only oma1-1 showed differences in activities and amounts of OXPHOS proteins. Among all the OXPHOS disorders found in oma1-1, the complex V deficiency is distinctive because it is mainly associated with decreased catalytic activity and not correlated with complex abundance, which has been observed in the case of supercomplex I + III2 and complex I deficiencies. Altogether, our study indicates that despite the presence of highly conservative homologs, the mitochondrial ATP-independent proteolytic system is not functionally conserved in plants as compared with yeast. Our findings also highlight the importance of AtOMA1 in maintenance of proper function of the OXPHOS system as well as in growth and development of Arabidopsis thaliana.

Transient Cell-intrinsic Activity Regulates the Migration and Laminar Positioning of Cortical Projection Neurons.

Neocortical microcircuits are built during development and require the coordinated assembly of excitatory glutamatergic projection neurons (PNs) into functional networks. Neuronal migration is an essential step in this process. In addition to cell-intrinsic mechanisms, external cues including neurotransmitters regulate cortical neuron migration, suggesting that early activity could influence this process. Here, we aimed to investigate the role of cell-intrinsic activity in migrating PNs in vivo using a designer receptor exclusively activated by a designer drug (DREADD) chemogenetic approach. In utero electroporation was used to specifically express the human M3 muscarinic cholinergic Gq-coupled receptor (hM3Dq) in PNs and calcium activity, migratory dynamics, gene expression, and laminar positioning of PNs were assessed following embryonic DREADD activation. We found that transient embryonic DREADD activation induced premature branching and transcriptional changes in migrating PNs leading to a persistent laminar mispositioning of superficial layer PNs into deep cortical layers without affecting expression of layer-specific molecular identity markers. In addition, live imaging approaches indicated that embryonic DREADD activation increased calcium transients in migrating PNs and altered their migratory dynamics by increasing their pausing time. Taken together, these results support the idea that increased cell-intrinsic activity during migration acts as a stop signal for migrating cortical PNs.

Lack of FTSH4 Protease Affects Protein Carbonylation, Mitochondrial Morphology, and Phospholipid Content in Mitochondria of Arabidopsis: New Insights into a Complex Interplay.

FTSH4 is one of the inner membrane-embedded ATP-dependent metalloproteases in mitochondria of Arabidopsis (Arabidopsis thaliana). In mutants impaired to express FTSH4, carbonylated proteins accumulated and leaf morphology was altered when grown under a short-day photoperiod, at 22°C, and a long-day photoperiod, at 30°C. To provide better insight into the function of FTSH4, we compared the mitochondrial proteomes and oxyproteomes of two ftsh4 mutants and wild-type plants grown under conditions inducing the phenotypic alterations. Numerous proteins from various submitochondrial compartments were observed to be carbonylated in the ftsh4 mutants, indicating a widespread oxidative stress. One of the reasons for the accumulation of carbonylated proteins in ftsh4 was the limited ATP-dependent proteolytic capacity of ftsh4 mitochondria, arising from insufficient ATP amount, probably as a result of an impaired oxidative phosphorylation (OXPHOS), especially complex V. In ftsh4, we further observed giant, spherical mitochondria coexisting among normal ones. Both effects, the increased number of abnormal mitochondria and the decreased stability/activity of the OXPHOS complexes, were probably caused by the lower amount of the mitochondrial membrane phospholipid cardiolipin. We postulate that the reduced cardiolipin content in ftsh4 mitochondria leads to perturbations within the OXPHOS complexes, generating more reactive oxygen species and less ATP, and to the deregulation of mitochondrial dynamics, causing in consequence the accumulation of oxidative damage.

[ATP-dependent proteases in the quality control of mitochondrial proteome]. / Proteazy zalezne od ATP w kontroli jakosci proteomu mitochondrialnego.

Mitochondria play the fundamental role in energy production and integration of many important metabolic and signalling pathways, which makes them essential for the function of a cell. The optimal operation of mitochondria depends on the qualitative and quantitative composition of the organellar proteins - the proteome. To maintain the homeostasis of the mitochondrial proteome, mitochondria developed a protein quality control system, which acts on the molecular, cellular and organellar levels. ATP-dependent proteases constitute a key element of this system. It consists of Lon/PIM1 and ClpXP proteases located in the mitochondrial matrix as well as AAA proteases anchored in the inner mitochondrial membrane. The ATP-dependent proteases degrade misfolded, damaged or not assembled proteins. These enzymes are also involved in complex regulatory mechanisms such as mitochondrial translation, fusion and response to stress. Lack of any of ATP-dependent proteases leads to mitochondrial dysfunction and the development of many major diseases in humans. This work summarizes the current knowledge of the ATP-dependent proteolytic system in mitochondria in different organisms.

Mitochondrial Proteome Studies in Seeds during Germination.

Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination is progressive development of structurally simple and metabolically quiescent promitochondria into fully active and cristae-containing mitochondria, known as mitochondrial biogenesis. This is a complex and tightly regulated process, which is accompanied by sequential and dynamic gene expression, protein synthesis, and post-translational modifications. The aim of this review is to give a comprehensive summary of seed mitochondrial proteome studies during germination of various plant model organisms. We describe different gel-based and gel-free proteomic approaches used to characterize mitochondrial proteomes of germinating seeds as well as challenges and limitations of these proteomic studies. Furthermore, the dynamic changes in the abundance of the mitochondrial proteomes of germinating seeds are illustrated, highlighting numerous mitochondrial proteins involved in respiration, tricarboxycylic acid (TCA) cycle, metabolism, import, and stress response as potentially important for seed germination. We then review seed mitochondrial protein carbonylation, phosphorylation, and S-nitrosylation as well as discuss the possible link between these post-translational modifications (PTMs) and the regulation of seed germination.

Serotonin Modulates Developmental Microglia via 5-HT2B Receptors: Potential Implication during Synaptic Refinement of Retinogeniculate Projections.

Maturation of functional neuronal circuits during central nervous system development relies on sophisticated mechanisms. First, axonal and dendritic growth should reach appropriate targets for correct synapse elaboration. Second, pruning and neuronal death are required to eliminate redundant or inappropriate neuronal connections. Serotonin, in addition to its role as a neurotransmitter, actively participates in postnatal establishment and refinement of brain wiring in mammals. Brain resident macrophages, that is, microglia, also play an important role in developmentally regulated neuronal death as well as in synaptic maturation and elimination. Here, we tested the hypothesis of cross-regulation between microglia and serotonin during postnatal brain development in a mouse model of synaptic refinement. We found expression of the serotonin 5-HT2B receptor on postnatal microglia, suggesting that serotonin could participate in temporal and spatial synchronization of microglial functions. Using two-photon microscopy, acute brain slices, and local delivery of serotonin, we observed that microglial processes moved rapidly toward the source of serotonin in Htr2B(+/+) mice, but not in Htr2B(-/-) mice lacking the 5-HT2B receptor. We then investigated whether some developmental steps known to be controlled by serotonin could potentially result from microglia sensitivity to serotonin. Using an in vivo model of synaptic refinement during early brain development, we investigated the maturation of the retinal projections to the thalamus and observed that Htr2B(-/-) mice present anatomical alterations of the ipsilateral projecting area of retinal axons into the thalamus. In addition, activation markers were upregulated in microglia from Htr2B(-/-) compared to control neonates, in the absence of apparent morphological modifications. These results support the hypothesis that serotonin interacts with microglial cells and these interactions participate in brain maturation.

Identification and characterization of high molecular weight complexes formed by matrix AAA proteases and prohibitins in mitochondria of Arabidopsis thaliana.

We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (approximately 2 MDa), similarly to their yeast counterparts. A smaller PHB complex (approximately 1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.

Plant mitochondria contain at least two i-AAA-like complexes.

The FtsH proteases, also called AAA proteases, are membrane-bound ATP-dependent metalloproteases. The Arabidopsis genome contains a total of 12 FtsH-like genes. Two of them, AtFtsH4 and AtFtsH11, encode proteins with a high similarity to Yme1p, a subunit of the i-AAA complex in yeast mitochondria. Phylogenetic analysis groups the AtFtsH4, AtFtsH11 and Yme1 proteins together, with AtFtsH4 being the most similar to Yme1. Using immunological method we demonstrate here that AtFtsH4 is an exclusively mitochondrial protein while AtFtsH11 is found in both chloroplasts and mitochondria. AtFtsH4 and AtFtsH11 proteases are integral parts of the inner mitochondrial membrane and expose their catalytic sites towards the intermembrane space, same as yeast i-AAA. Database searches revealed that orthologs of AtFtsH4 and AtFtsH11 are present in both monocotyledonous and dicotyledonous plants. The two plant i-AAA proteases differ significantly in their termini: the FtsH4 proteins have a characteristic alanine stretch at the C-terminal end while FtsH11s have long N-terminal extensions. Blue-native gel electrophoresis revealed that AtFtsH4 and AtFtsH11 form at least two complexes with apparent molecular masses of about 1500 kDa. This finding implies that plants, in contrast to fungi and metazoa, have more than one complex with a topology similar to that of yeast i-AAA.

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning, localization, and putative function.

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.