Distinct PSD-95 clusters are primary landmarks of postsynaptic densities (PSDs), which are specialized membrane regions for synapses. However, the mechanism that defines the locations of PSD-95 clusters and whether or how they are reorganized inside individual dendritic spines remains controversial. Because palmitoylation regulates PSD-95 membrane targeting, we combined a conformation-specific recombinant antibody against palmitoylated PSD-95 with live-cell super-resolution imaging and discovered subsynaptic nanodomains composed of palmitoylated PSD-95 that serve as elementary units of the PSD. PSD-95 in nanodomains underwent continuous de/repalmitoylation cycles driven by local palmitoylating activity, ensuring the maintenance of compartmentalized PSD-95 clusters within individual spines. Plasma membrane targeting of DHHC2 palmitoyltransferase rapidly recruited PSD-95 to the plasma membrane and proved essential for postsynaptic nanodomain formation. Furthermore, changes in synaptic activity rapidly reorganized PSD-95 nano-architecture through plasma membrane-inserted DHHC2. Thus, the first genetically encoded antibody sensitive to palmitoylation reveals an instructive role of local palmitoylation machinery in creating activity-responsive PSD-95 nanodomains, contributing to the PSD (re)organization.

The unusually high demand for metals in the brain, along with insufficient understanding of how their dysregulation contributes to neurological diseases, motivates the study of how inorganic chemistry influences neural circuitry. We now report that the transition metal copper is essential for regulating rest-activity cycles and arousal. Copper imaging and gene expression analysis in zebrafish identifies the locus coeruleus-norepinephrine (LC-NE) system, a vertebrate-specific neuromodulatory circuit critical for regulating sleep, arousal, attention, memory and emotion, as a copper-enriched unit with high levels of copper transporters CTR1 and ATP7A and the copper enzyme dopamine β-hydroxylase (DBH) that produces NE. Copper deficiency induced by genetic disruption of ATP7A, which loads copper into DBH, lowers NE levels and hinders LC function as manifested by disruption in rest-activity modulation. Moreover, LC dysfunction caused by copper deficiency from ATP7A disruption can be rescued by restoring synaptic levels of NE, establishing a molecular CTR1-ATP7A-DBH-NE axis for copper-dependent LC function.

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

We investigated whether low intensity dawn-dusk simulation (DDS), a 'naturalistic' form of light therapy designed to embed sleep in its accustomed phase, could improve the disturbed circadian rest-activity cycle, nocturnal sleep and and/or cognitive functions in dementia. A protocol of 3 weeks each of baseline, treatment and follow-up was completed by 13 patients (85yr old+/-5yr, MMSE 14+/-5; n=9 DDS versus n=4 'placebo' dim red light) who wore an activity/lux monitor throughout. There were no significant changes in clinical or cognitive status, nor modification of circadian stability or amplitude characteristics of the rest-activity cycle. However, two aspects of sleep responded to DDS but not to dim red light. The main sleep episode was 1:14h earlier during treatment (p=0.03) compared with before and after DDS. With respect to actimetry-determined sleep variables, the DDS group tended to have shortened 'sleep latency', longer 'sleep duration', more nocturnal immobility and less nocturnal activity than the dim red group (p<0.1). In parallel, nighttime light exposure tended to be reduced (p=0.07). These promising findings-after only 3 weeks of light treatment in elderly patients with advanced dementia-suggest that the circadian timing system remains functionally responsive even to low intensity DDS light. Increasing zeitgeber strength is an important strategy for improving sleep quality and timing in dementia, and DDS light therapy may provide one of the appropriate means to do so.

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8-24.9 h. After having verified that the light-dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

Inflammation, oxidative stress, and protease-antiprotease imbalance have been suggested to be a pathogenic triad in chronic obstructive pulmonary disease (COPD). However, it is not clear how proteases interact with components of inflammatory pathways. Therefore, this study aimed to evaluate the effect of neutrophil elastase (NE) on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production and determine the molecular mechanism in human bronchial epithelial cells (HBECs). Immortalized bronchial epithelial cells and primary HBECs were used to investigate the impact of NE on LPS-induced IL-8 production. The molecular mechanism by which NE modulated LPS-induced IL-8 production was confirmed in elastase-treated C57BL/6 mice and primary HBECs obtained from COPD patients and healthy controls. The results showed that NE treatment synergistically augmented LPS-induced IL-8 production in both immortalized bronchial epithelial cells and primary HBECs. NE partially degraded peroxisome proliferator-activated receptor gamma (PPARγ), which is known to regulate IL-8 production in the nucleus. Treatment with a PPARγ agonist and overexpression of PPARγ reversed the NE-induced synergistic increase in LPS-induced IL-8 production. Moreover, PPARγ levels were lower in lung homogenates and lung epithelial cells from elastase-treated mice than in those from saline-treated mice. In accordance with the findings in mice, PPARγ levels were lower in primary HBECs from COPD patients than in those from healthy never-smokers or healthy smokers. In conclusion, a vicious cycle of mutual augmentation of protease activity and inflammation resulting from PPARγ degradation plays a role in the pathogenesis of COPD.

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.

Genome-wide transcript profiling and analyses of enzyme activities from central carbon and nitrogen metabolism show that transcript levels undergo marked and rapid changes during diurnal cycles and after transfer to darkness, whereas changes in activities are smaller and delayed. In the starchless pgm mutant, where sugars are depleted every night, there are accentuated diurnal changes in transcript levels. Enzyme activities in this mutant do not show larger diurnal changes; instead, they shift towards the levels found in the wild type after several days of darkness. This indicates that enzyme activities change slowly, integrating the changes in transcript levels over several diurnal cycles.

Organic soilless production in containers requires substrates with appropriate physicochemical and biological properties to ensure that production is sustainable and profitable for several production cycles. The main objective of this study was to comprehensively evaluate these properties in three different mixtures of organic substrates (vermicompost [V] and coconut fibers [CF] in ratios 20V80CF, 40V60CF, 60V40CF) for four horticultural crop production cycles (PCs) using vermicompost tea (VT) as the main source of nutrients.

We have previously reported that rats that experienced 3 h of daily maternal separation during the first 2 weeks of birth (MS) showed binge-like eating behaviors with increased activity of the hypothalamic-pituitary-adrenal axis when they were subjected to fasting/refeeding cycles repeatedly. In this study, we have examined the psychoemotional behaviors of MS rats on the fasting/refeeding cycles, together with their brain dopamine levels. Fasting/refeeding cycles normalized the ambulatory activity of MS rats, which was decreased by MS experience. Depression-like behaviors, but not anxiety, by MS experience were improved after fasting/refeeding cycles. Fasting/refeeding cycles did not significantly affect the behavioral scores of nonhandled (NH) control rats. Fasting/refeeding cycles increased dopamine levels not only in the hippocampus but also in the midbrain dopaminergic neurons in MS rats, but not in NH controls. Results demonstrate that fasting/refeeding cycles increase the mesohippocampal dopaminergic activity and improve depression-like behaviors in rats that experienced MS. Together with our previous paper, it is suggested that increased dopamine neurotransmission in the hippocampus may be implicated in the underlying mechanisms by which the fasting/refeeding cycles induce binge-like eating and improve depression-like behaviors in MS rats.

Heterotrimeric G proteins, composed of Gα, Gβ, and Gγ subunits, are a class of signal transduction complexes with broad roles in human health and agriculturally relevant plant physiological and developmental traits. In the classic paradigm, guanine nucleotide binding to the Gα subunit regulates the activation status of the complex. We sought to develop improved methods for heterologous expression and rapid purification of Gα subunits. Using GPA1, the sole canonical Gα subunit of the model plant species, Arabidopsis thaliana, we observed that, compared to conventional purification methods, rapid StrepII-tag mediated purification facilitates isolation of protein with increased GTP binding and hydrolysis activities. Human GNAI1 purified using our approach also displayed the expected binding and hydrolysis activities, indicating our protocol is applicable to mammalian Gα subunits, potentially including those for which purification of enzymatically active protein has been historically problematic. We subsequently utilized domain swaps of GPA1 and human GNAO1 to demonstrate that the inherent instability of GPA1 is a function of the interaction between the Ras and helical domains. Additionally, we found that GPA1-GNAO1 domain swaps partially uncouple the instability from the rapid nucleotide binding kinetics displayed by GPA1.

Pt-loaded anatase TiO2 (Pt/TiO2-A) was found to be a highly active and stable catalyst for SO3 decomposition at moderate temperatures (∼600 °C), which will prove to be the key for solar thermochemical water-splitting processes used to produce H2. The catalytic activity of Pt/TiO2-A was found to be markedly superior to that of a Pt catalyst supported on rutile TiO2 (Pt/TiO2-R), which has been extensively studied at a higher reaction temperature range (≥800 °C); this superior activity was found despite the two being tested with similar surface areas and metal dispersions after the catalytic reactions. The higher activity of Pt on anatase is in accordance with the abundance of metallic Pt (Pt0) found for this catalyst, which favors the dissociative adsorption of SO3 and the fast removal of the products (SO2 and O2) from the surface. Conversely, Pt was easily oxidized to the much less active PtO2 (Pt4+), with the strong interactions between the oxide and rutile TiO2 forming a fully coherent interface that limited the active sites. A long-term stability test of Pt/TiO2-A conducted for 1000 h at 600 °C demonstrated that there was no indication of noticeable deactivation (activity loss ≤ 4%) over the time period; this was because the phase transformation from anatase to rutile was completely prevented. The small amount of deactivation that occurred was due to the sintering of Pt and TiO2 and the loss of Pt under the harsh reaction atmosphere.

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. In this study, we use integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). Our evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2F1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, our results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.

We demonstrated carbon-neutral (CN) energy circulation using glycolic acid (GC)/oxalic acid (OX) redox couple. Here, we report fundamental studies on both catalyst search for power generation process, i.e. GC oxidation, and elemental steps for fuel generation process, i.e. OX reduction, in CN cycle. The catalytic activity test on various transition metals revealed that Rh, Pd, Ir, and Pt have preferable features as a catalyst for electrochemical oxidation of GC. A carbon-supported Pt catalyst in alkaline conditions exhibited higher activity, durability, and product selectivity for electrooxidation of GC rather than those in acidic media. The kinetic study on OX reduction clearly indicated that OX reduction undergoes successive two-electron reductions to form GC. Furthermore, application of TiO2 catalysts with large specific area for electrochemical reduction of OX facilitates the selective formation of GC.

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. We observed that timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

This meta-analysis was aimed to evaluate the association between maternal physical activity before IVF/ICSI cycles and reproductive outcomes.

Ssr4 serves as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but remains functionally uncharacterized due to its essentiality for yeast viability. Here, we report pleiotropic effects of the deletion of the ssr4 ortholog nonessential for cell viability in Beauveria bassiana, an asexual insect mycopathogen. The deletion of ssr4 resulted in severe growth defects on different carbon/nitrogen sources, increased hyphal hydrophilicity, blocked hyphal differentiation, and 98% reduced conidiation capacity compared to a wild-type standard. The limited Δssr4 conidia featured an impaired coat with disordered or obscure hydrophobin rodlet bundles, decreased hydrophobicity, increased size, and lost insect pathogenicity via normal cuticle infection and 90% of virulence via intrahemocoel injection. The expression of genes required for hydrophobin biosynthesis and assembly of the rodlet layer was drastically repressed in more hydrophilic Δssr4 cells. Transcriptomic analysis revealed 2,517 genes differentially expressed in the Δssr4 mutant, including 1,505 downregulated genes and 1,012 upregulated genes. The proteins encoded by hundreds of repressed genes were involved in metabolism and/or transport of carbohydrates, amino acids, and lipids, inorganic ion transport and energy production or conversion, including dozens involved in DNA replication, transcription, translation, and posttranslational modifications. However, purified Ssr4 samples showed no DNA-binding activity, implying that the role of Ssr4 in genome-wide gene regulation could rely upon its acting as a cosubunit of the two complexes. These findings provide the first insight into an essential role of Ssr4 in the asexual cycle in vitro and in vivo of B. bassiana and highlights its importance for the filamentous fungal lifestyle.IMPORTANCE Ssr4 is known to serve as a cosubunit of chromatin-remodeling SWI/SNF and RSC complexes in yeasts but has not been functionally characterized in fungi. This study unveils for the first time the pleiotropic effects caused by deletion of ssr4 and its role in mediating global gene expression in a fungal insect pathogen. Our findings confirm an essential role of Ssr4 in hydrophobin biosynthesis and assembly required for growth, differentiation, and development of aerial hyphae for conidiation and conidial adhesion to insect surface and its essentiality for insect pathogenicity and virulence-related cellular events. Importantly, Ssr4 can regulate nearly one-fourth of all genes in the fungal genome in direct and indirect manners, including dozens involved in gene activity and hundreds involved in metabolism and/or transport of carbohydrates, amino acids, lipids, and/or inorganic ions. These findings highlight a significance of Ssr4 for filamentous fungal lifestyle.

Cerebral metabolism varies dramatically as a function of sleep state. Brain concentration of lactate, the end product of glucose utilization via glycolysis, varies as a function of sleep state, and like slow wave activity (SWA) in the electroencephalogram (EEG), increases as a function of time spent awake or in rapid eye movement sleep and declines as a function of time spent in slow wave sleep (SWS). We sought to determine whether lactate concentration exhibits homeostatic dynamics akin to those of SWA in SWS. Lactate concentration in the cerebral cortex was measured by indwelling enzymatic biosensors. A set of equations based conceptually on Process S (previously used to quantify the homeostatic dynamics of SWA) was used to predict the sleep/wake state-dependent dynamics of lactate concentration in the cerebral cortex. Additionally, we applied an iterative parameter space-restricting algorithm (the Nelder-Mead method) to reduce computational time to find the optimal values of the free parameters. Compared to an exhaustive search, this algorithm reduced the computation time required by orders of magnitude. We show that state-dependent lactate concentration dynamics can be described by a homeostatic model, but that the optimal time constants for describing lactate dynamics are much smaller than those of SWA. This disconnect between lactate dynamics and SWA dynamics does not support the concept that lactate concentration is a biochemical mediator of sleep homeostasis. However, lactate synthesis in the cerebral cortex may nonetheless be informative with regard to sleep function, since the impact of glycolysis on sleep slow wave regulation is only just now being investigated.