Developing a High-Umami, Low-Salt Soy Sauce through Accelerated Moromi Fermentation with Corynebacterium and Lactiplantibacillus Strains.

The traditional fermentation process of soy sauce employs a hyperhaline model and has a long fermentation period. A hyperhaline model can improve fermentation speed, but easily leads to the contamination of miscellaneous bacteria and fermentation failure. In this study, after the conventional koji and moromi fermentation, the fermentation broth was pasteurized and diluted, and then inoculated with three selected microorganisms including Corynebacterium glutamicum, Corynebacterium ammoniagenes, and Lactiplantibacillus plantarum for secondary fermentation. During this ten-day fermentation, the pH, free amino acids, organic acids, nucleotide acids, fatty acids, and volatile compounds were analyzed. The fermentation group inoculated with C. glutamicum accumulated the high content of amino acid nitrogen of 0.92 g/100 mL and glutamic acid of 509.4 mg/100 mL. The C. ammoniagenes group and L. plantarum group were rich in nucleotide and organic acid, respectively. The fermentation group inoculated with three microorganisms exhibited the best sensory attributes, showing the potential to develop a suitable fermentation method. The brewing speed of the proposed process in this study was faster than that of the traditional method, and the umami substances could be significantly accumulated in this low-salt fermented model (7% w/v NaCl). This study provides a reference for the low-salt and rapid fermentation of seasoning.

Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons.

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

The AD odyssey 2023: Tales of single cell.

Deciphering cellular changes in Alzheimer's disease (AD) using large cohorts with defined clinical stages is essential for understanding the diverse trajectories of AD progression. In this issue of Cell, five studies harnessed the power of single-nuclei RNA sequencing (snRNA-seq) and single-nuclei ATAC sequencing (snATAC-seq) at unprecedented scale and revealed exciting insights into cell-type-specific mechanisms underlying the progression of AD pathogenesis.

Deletion of Small GTPase H-Ras Rescues Memory Deficits and Reduces Amyloid Plaque-Associated Dendritic Spine Loss in Transgenic Alzheimer's Mice.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder, affecting millions of lives without a cure. While the molecular mechanism of AD remains obscure, emerging evidence suggests that small GTPases, a group of GTP-binding proteins that regulate a plethora of essential cellular events, modulate the pathogenic process of AD. Among those, the small GTPase H-Ras, extensively studied in cancer, regulates synaptic function, and both upstream and downstream signaling pathways of H-Ras have been implicated in AD. However, the role of H-Ras per se in AD pathogenesis had not been explored previously. In the present study, the impact of Hras deletion on cognitive function and amyloid pathology was investigated in transgenic APP/PS1 mice of AD. Behavioral assessments showed that the absence of Hras rescued spatial memory deficit in APP/PS1 mice at 9 months of age. The pathological evaluation demonstrated that Hras deletion reduced cortical amyloid deposition and astrogliosis. Furthermore, Hras deficiency protected against amyloid plaque-associated loss of dendritic spines in APP/PS1 mice. Intriguingly, canonical signaling pathways downstream of H-Ras were not affected by the absence of Hras in the brain. Unbiased transcriptomic analysis revealed that lack of H-Ras affected the expression of select genes in the brain of AD mice and identified a novel connection between H-Ras and Annexin A4, a calcium-dependent phospholipid-binding protein that has been shown to regulate membrane repair, neuroinflammation, and calcium homeostasis. Taken together, these data indicate that H-Ras modifies the pathogenic process of AD and may serve as a potential therapeutic target for AD.

Microglial TREM2 at the Intersection of Brain Aging and Alzheimer's Disease.

As resident immune cells of the brain, microglia serve pivotal roles in regulating neuronal function under both physiological and pathological conditions, including aging and the most prevalent neurodegenerative disease, Alzheimer's disease (AD). Instructed by neurons, microglia regulate synaptic function and guard brain homeostasis throughout life. Dysregulation of microglial function, however, can lead to dire consequences, including aggravated cognitive decline during aging and exacerbated neuropathology in diseases. The triggering receptor expressed on myeloid cells 2 (TREM2) is a key regulator of microglial function. Loss-of-function variants of TREM2 are associated with an increased risk of AD. TREM2 orchestrates the switch of microglial transcriptome programming that modulates microglial chemotaxis, phagocytosis, and inflammatory responses, as well as microglial regulation of synaptic function in health and disease. Intriguingly, the outcome of microglial/TREM2 function is influenced by age and the context of neuropathology. This review summarizes the rapidly growing research on TREM2 under physiological conditions and in AD, particularly highlighting the impact of TREM2 on neuronal function.

Molecular Insights into Cell Type-specific Roles in Alzheimer's Disease: Human Induced Pluripotent Stem Cell-based Disease Modelling.

Alzheimer's disease (AD) is the most common cause of dementia resulting in widespread degeneration of the central nervous system with severe cognitive impairment. Despite the devastating toll of AD, the incomplete understanding of the complex molecular mechanisms hinders the expeditious development of effective cures. Emerging evidence from animal studies has shown that different brain cell types play distinct roles in the pathogenesis of AD. Glutamatergic neurons are preferentially affected in AD and pronounced gliosis contributes to the progression of AD in both a cell-autonomous and a non-cell-autonomous manner. Much has been discovered through genetically modified animal models, yet frequently failed translational attempts to clinical applications call for better disease models. Emerging evidence supports the significance of human-induced pluripotent stem cell (iPSC) derived brain cells in modeling disease development and progression, opening new avenues for the discovery of molecular mechanisms. This review summarizes the function of different cell types in the pathogenesis of AD, such as neurons, microglia, and astrocytes, and recognizes the potential of utilizing the rapidly growing iPSC technology in modeling AD.

LRP8, modulated by miR-1262, promotes tumour progression and forecasts the prognosis of patients in breast cancer.

This research was designed to detect the function of low-density lipoprotein receptor (LDLR)-related protein 8 (LRP8) in breast cancer (BC). Our results revealed that LRP8 was highly expressed in BC tissues and cell lines compared with human normal breast tissues. The poor prognosis of patients with BC was associated with the up-regulation of LRP8 while inversely connected with overexpression of miR-1262. Functionally, LRP8 depletion in BC cells impaired the proliferative, clonogenic, invasive, and migratory capabilities, which was consistent with the effects of upregulated miR-1262. Bioinformatics prediction and luciferase reporter assay confirmed that miR-1262 was an upstream factor for LRP8 and negatively regulated the expression of LRP8. Further experiments illustrated that the co-transfection of miR-1262 antamir and si-LRP8 could significantly suppress the promoting impacts caused by the transfection of miR-1262 antamir alone. These findings highlighted that LRP8 accelerated the BC development by contributing cellular aggressiveness, which was modulated by miR-1262.

Effects of Glucose Oxidase Supplementation on the Growth Performance, Antioxidative and Inflammatory Status, Gut Function, and Microbiota Composition of Broilers Fed Moldy Corn.

BACKGROUND: Glucose oxidase is widely used as a livestock feed additive owing to its beneficial effects on growth performance and antioxidant activity. However, little is known about the effects of the enzyme on intestinal health. METHODS: To investigate the effects of glucose oxidase supplementation on the growth performance, intestinal function, and microbiota composition of broilers fed moldy corn, newly hatched Arbor Acres broilers were each randomly assigned to one of four groups, which were fed a basal diet (CON), a contaminated diet (10% moldy corn) (MC), a basal diet supplemented with 0.01% glucose oxidase (GOD), or a contaminated diet supplemented with 0.01% glucose oxidase (MCG). RESULTS: We found that the average weight gain (ADG) of the MC group was significantly lower than those of the CON and GOD groups, and there were no significant differences in ADG between the MCG group and the CON and GOD groups. Intestinal morphology results revealed irregularly arranged villi and microvilli in the ilea from the MC group, whereas those from the other three groups were aligned regularly. Tight-junction protein analysis showed that both ZO-1 expression and claudin-4 expression in the MC group were significantly lower than those in the other groups. Inflammation cytokines analysis showed lower serum concentration of interleukin-10, as well as its mRNA expression in the ileum of the MC group, when compared with those of the other groups. Additionally, we observed lower glutathione peroxidase and total superoxide dismutase activity and higher malonaldehyde concentration in the MC group than those in the MCG group. The α and ß diversity of microbiota profiling indicated that the cecal microbiota in the MC group differed from those in the other three groups. CONCLUSION: The results indicated that glucose oxidase supplementation was able to prevent the adverse effects from mycotoxin exposure on growth performance, antioxidant activity, inflammatory response, intestinal function, and microbiota composition in broilers. We suggested that glucose oxidase supplementation can be used in broilers to mitigate the adverse effects of moldy feed, and its benefits are due to its effect on intestinal microbiota composition.

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells.

Protein prenylation involves the attachment of one or two isoprenoid group(s) onto cysteine residues positioned near the C-terminus. This modification is essential for many signal transduction processes. In this work, the use of the probe C15AlkOPP for metabolic labeling and identification of prenylated proteins in a variety of cell lines and primary cells is explored. Using a single isoprenoid analogue, 78 prenylated protein groups from the three classes of prenylation substrates were identified including three novel prenylation substrates in a single experiment. Applying this method to three brain-related cell lines including neurons, microglia, and astrocytes showed substantial overlap (25%) in the prenylated proteins identified. In addition, some unique prenylated proteins were identified in each type. Eight proteins were observed exclusively in neurons, five were observed exclusively in astrocytes and three were observed exclusively in microglia, suggesting their unique roles in these cells. Furthermore, inhibition of farnesylation in primary astrocytes revealed the differential responses of farnesylated proteins to an FTI. Importantly, these results provide a list of 19 prenylated proteins common to all the cell lines studied here that can be monitored using the C15AlkOPP probe as well as a number of proteins that were observed in only certain cell lines. Taken together, these results suggest that this chemical proteomic approach should be useful in monitoring the levels and exploring the underlying role(s) of prenylated proteins in various diseases.

Neuronal Protein Farnesylation Regulates Hippocampal Synaptic Plasticity and Cognitive Function.

Protein prenylation is a post-translational lipid modification that governs a variety of important cellular signaling pathways, including those regulating synaptic functions and cognition in the nervous system. Two enzymes, farnesyltransferase (FT) and geranylgeranyltransferase type I (GGT), are essential for the prenylation process. Genetic reduction of FT or GGT ameliorates neuropathology but only FT haplodeficiency rescues cognitive function in transgenic mice of Alzheimer's disease. A follow-up study showed that systemic or forebrain neuron-specific deficiency of GGT leads to synaptic and cognitive deficits under physiological conditions. Whether FT plays different roles in shaping neuronal functions and cognition remains elusive. This study shows that in contrast to the detrimental effects of GGT reduction, systemic haplodeficiency of FT has little to no impact on hippocampal synaptic plasticity and cognition. However, forebrain neuron-specific FT deletion also leads to reduced synaptic plasticity, memory retention, and hippocampal dendritic spine density. Furthermore, a novel prenylomic analysis identifies distinct pools of prenylated proteins that are affected in the brain of forebrain neuron-specific FT and GGT knockout mice, respectively. Taken together, this study uncovers that physiological levels of FT and GGT in neurons are essential for normal synaptic/cognitive functions and that the prenylation status of specific signaling molecules regulates neuronal functions.

Loss of TREM2 Confers Resilience to Synaptic and Cognitive Impairment in Aged Mice.

Triggering receptor expressed on myeloid cells 2 (TREM2), a receptor exclusively expressed by microglia in the brain, modulates microglial immune homeostasis. Human genetic studies have shown that the loss-of-function mutations in TREM2 signaling are strongly associated with an elevated risk of age-related neurodegenerative diseases including Alzheimer's disease (AD). Numerous studies have investigated the impact of TREM2 deficiency in the pathogenic process of AD. However, the role of TREM2 in shaping neuronal and cognitive function during normal aging is underexplored. In the present study, we employed behavioral, electrophysiological, and biochemical approaches to assess cognitive and synaptic function in male and female young and aged TREM2-deficient (Trem2-/-) mice compared with age-matched, sex-matched, and genetic background-matched wild-type (WT) C57BL/6J controls. Young Trem2-/- mice exhibited normal cognitive function and synaptic plasticity but had increased dendritic spine density compared with young WT. Unexpectedly, aged Trem2-/- mice showed superior cognitive performance compared with aged WT controls. Consistent with the behavioral data, aged Trem2-/- mice displayed significantly enhanced hippocampal long-term potentiation (LTP) and increased dendritic spine density and synaptic markers compared with aged WT mice. Taken together, these findings suggest that loss of TREM2 affects the neuronal structure and confers resilience to age-related synaptic and cognitive impairment during non-pathogenic aging.SIGNIFICANCE STATEMENT Microglia are innate immune cells of the brain that orchestrates neurodevelopment, synaptic function, and immune response to environmental stimuli. Microglial triggering receptor expressed on myeloid cells 2 (TREM2) signaling plays pivotal roles in regulating these functions and loss of TREM2 signaling leads to increased risk of developing age-related neurologic disorders. However, the neurologic role of TREM2 in normal aging is poorly understood. The results of the present study unveil the positive impacts of TREM2 deficiency on cognitive and synaptic function during aging and suggest that TREM2 may exert detrimental effects on neuronal function. The possibility of age-related negative impacts from TREM2 is critically important since TREM2 has emerged as a major therapeutic target for Alzheimer's dementia.

Peripheral versus central nervous system APOE in Alzheimer's disease: Interplay across the blood-brain barrier.

The apolipoprotein E (APOE) ε4 allele has been demonstrated as the preeminent genetic risk factor for late onset Alzheimer's disease (AD), which comprises greater than 90% of all AD cases. The discovery of the connection between different APOE genotypes and AD risk in the early 1990s spurred three decades of intense and comprehensive research into the function of APOE in the normal and diseased brain. The importance of APOE in the periphery has been well established, due to its pivotal role in maintaining cholesterol homeostasis and cardiovascular health. The influence of vascular factors on brain function and AD risk has been extensively studied in recent years. As a major apolipoprotein regulating multiple molecular pathways beyond its canonical lipid-related functions in the periphery and the central nervous system, APOE represents a critical link between the two compartments, and may influence AD risk from both sides of the blood-brain barrier. This review discusses recent advances in understanding the different functions of APOE in the periphery and in the brain, and highlights several promising APOE-targeted therapeutic strategies for AD.

Astroglia contribute to the pathogenesis of spinocerebellar ataxia Type 1 (SCA1) in a biphasic, stage-of-disease specific manner.

Spinocerebellar ataxia type 1 (SCA1) is a fatal, dominantly inherited neurodegenerative disease caused by the expansion of CAG repeats in the Ataxin-1 (ATXN1) gene. SCA1 is characterized by balance and coordination deficits due to the predominant loss of Purkinje neurons in the cerebellum. We previously demonstrated that cerebellar astrogliosis beings during the early stages of SCA1, prior to onset of motor deficits and loss of Purkinje neurons. We communicate here that cerebellar astrogliosis contributes to SCA1 pathogenesis in a biphasic, stage of disease dependent manner. We modulated astrogliosis by selectively reducing pro-inflammatory transcriptional regulator nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling in astroglia via a Cre-lox mouse genetic approach. Our results indicate that inhibition of astroglial NF-κB signaling, prior to motor deficit onset, exacerbates disease severity. This is suggestive of a neuroprotective role mediated by astroglia during early stage SCA1. In contrast, inhibition of astroglial NF-κB signaling during late stage of disease ameliorated motor deficits, indicating a potentially harmful role of astroglia late in SCA1. These results indicate that astrogliosis may have a critical and dual role in disease. If so, our results imply that anti-inflammatory astroglia-based therapeutic approaches may need to consider disease progression to achieve therapeutic efficacy.

Inhibition of NF-κB signaling in IKKßF/F;LysM Cre mice causes motor deficits but does not alter pathogenesis of Spinocerebellar ataxia type 1.

Spinocerebellar Ataxia type 1 (SCA1) is a fatal neurodegenerative genetic disease that is characterized by pronounced neuronal loss and gliosis in the cerebellum. We have previously demonstrated microglial activation, measured as an increase in microglial density in cerebellar cortex and an increase in the production of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), in the cerebellum of the ATXN1[82Q] transgenic mouse model of SCA1. To examine the role of activated state of microglia in SCA1, we used a Cre-Lox approach with IKKßF/F;LysM Cre mice intended to reduce inflammatory NF-κB signaling, selectively in microglia. ATXN1[82Q];IKKßF/F;LysM Cre mice showed reduced cerebellar microglial density and production of TNFα compared to ATXN1[82Q] mice, yet reducing NF-κB did not ameliorate motor impairments and cerebellar cellular pathologies. Unexpectedly, at 12 weeks of age, control IKKßF/F;LysM Cre mice showed motor deficits equal to ATXN1[82Q] mice that were dissociated from any obvious neurodegenerative changes in the cerebellum, but were rather associated with a developmental impairment that presented as a retention of climbing fiber synaptic terminals on the soma of Purkinje neurons. These results indicate that NF-κB signaling is required for increase in microglial numbers and TNF-α production in the cerebella of ATXN1[82Q] mouse model of SCA1. Furthermore, these results elucidate a novel role of canonical NF-κB signaling in pruning of surplus synapses on Purkinje neurons in the cerebellum during development.

Inhibition of colony-stimulating factor 1 receptor early in disease ameliorates motor deficits in SCA1 mice.

BACKGROUND: Polyglutamine (polyQ) expansion in the protein Ataxin-1 (ATXN1) causes spinocerebellar ataxia type 1 (SCA1), a fatal dominantly inherited neurodegenerative disease characterized by motor deficits, cerebellar neurodegeneration, and gliosis. Currently, there are no treatments available to delay or ameliorate SCA1. We have examined the effect of depleting microglia during the early stage of disease by using PLX, an inhibitor of colony-stimulating factor 1 receptor (CSFR1), on disease severity in a mouse model of SCA1. METHODS: Transgenic mouse model of SCA1, ATXN1[82Q] mice, and wild-type littermate controls were treated with PLX from 3 weeks of age. The effects of PLX on microglial density, astrogliosis, motor behavior, atrophy, and gene expression of Purkinje neurons were examined at 3 months of age. RESULTS: PLX treatment resulted in the elimination of 70-80% of microglia from the cerebellum of both wild-type and ATXN1[82Q] mice. Importantly, PLX ameliorated motor deficits in SCA1 mice. While we have not observed significant improvement in the atrophy or disease-associated gene expression changes in Purkinje neurons upon PLX treatment, we have detected reduced expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) and increase in the protein levels of wild-type ataxin-1 and post-synaptic density protein 95 (PSD95) that may help improve PN function. CONCLUSIONS: A decrease in the number of microglia during an early stage of disease resulted in the amelioration of motor deficits in SCA1 mice.

Converting biowaste corncob residue into high value added porous carbon for supercapacitor electrodes.

In this report, corncob residue, the main by-product in the furfural industry, is used as a precursor to prepare porous carbon by a simple and direct thermal treatment: one-step activation without pre-carbonization. As a consequence, the corncob residue derived porous carbon achieves a high surface area of 1210 m(2) g(-1) after ash-removal. The carbon material has the advantages of low cost and low environmental impact, with a superior electrochemical performance compared to those polymer-based synthetic carbons as electrode material for a supercapacitor. The carbon electrode exhibits a high capacitance of 314 F g(-1) in 6M KOH electrolyte. The corresponding sample also shows a superb cycling stability. Almost no capacitance decay was observed after 100,000 cycles. The excellent electrochemical performance is due to the combination of a high specific surface area with a fraction of mesopores and highly stable structure.

Diaminohexane-assisted preparation of coral-like, poly(benzoxazine)-based porous carbons for electrochemical energy storage.

The assembly of commercial silica colloids in the presence of 1,6-diaminohexane and their subsequent encapsulation by poly(benzoxazine) have been used to produce coral-like porous carbons. The pyrolysis of the polymer followed by the removal of the silica produces a carbon with a continuous skeleton that contains spherical medium-size pores as "reservoirs" with a structure similar to a bunch of grapes. The total volume and the diameter of the "reservoir" pores are tunable. The coral-like morphology and the pore structure of the carbons make them suitable for use as electrode materials for supercapacitors and lithium-ion batteries. As supercapacitor electrodes, they exhibit excellent long-term cycle stability (almost no capacitance fading after 20,000 cycles at a current density of 1 A g(-1)) and good rate capability with capacitance retention of 88% (from 0.1 A g(-1) to 5 A g(-1)). Meanwhile, as a matrix for the encapsulation of SnO2 nanoparticles for Li-ion storage, the electrodes also show a high specific capacity and good cycling stability, i.e., 900 mA h g(-1) after 50 charge-discharge cycles. The good electrochemical performance of such carbons shows that they are promising candidate electrode materials for electrochemical energy storage.

Atorvastatin prevents amyloid-ß peptide oligomer-induced synaptotoxicity and memory dysfunction in rats through a p38 MAPK-dependent pathway.

AIM: To investigate whether atorvastatin treatment could prevent Aß1-42 oligomer (AßO)-induced synaptotoxicity and memory dysfunction in rats, and to elucidate the mechanisms involved in the neuroprotective actions of atorvastatin. METHODS: SD rats were injected with AßOs (5 nmol, icv). The rats were administrated with atorvastatin (10 mg·kg(-1)·d(-1), po) for 2 consecutive weeks (the first dose was given 5 d before AßOs injection). The memory impairments were evaluated with Morris water maze task. The expression of inflammatory cytokines in the hippocampus was determined using ELISA assays. The levels of PSD-95 and p38MAPK proteins in rat hippocampus were evaluated using Western blot analysis. For in vitro experiments, cultured rat hippocampal neurons were treated with AßOs (50 nmol/L) for 48 h. The expression of MAP-2 and synaptophysin in the neurons was detected with immunofluorescence. RESULTS: The AßO-treated rats displayed severe memory impairments in Morris water maze tests, and markedly reduced levels of synaptic proteins synaptophysin and PSD-95, increased levels of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and p38MAPK activation in the hippocampus. All these effects were prevented or substantially attenuated by atorvastatin administration. Pretreatment of cultured hippocampal neurons with atorvastatin (1 and 5 µmol/L) concentration-dependently attenuated the AßO-induced synaptotoxicity, including the loss of dendritic marker MAP-2, and synaptic proteins synaptophysin and PSD-95. Pretreatment of the cultured hippocampal neurons with the p38MAPK inhibitor SB203580 (5 µmol/L) blocked the AßO-induced loss of synaptophysin and PSD-95. CONCLUSION: Atorvastatin prevents AßO-induced synaptotoxicity and memory dysfunction through a p38MAPK-dependent pathway.

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3ß signaling pathways.

AIM: To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect. METHODS: Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05-10 µmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3ß (GSK-3ß) in the cortical neurons were evaluated using Western blotting analyses. RESULTS: Atorvastatin (0.05-10 µmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 µmol/L) and wortmannin (5 µmol/L), Akt inhibitor tricribine (1 µmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 µmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 µmol/L). Moreover, atorvastatin (10 µmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 µmol/L) significantly increased the phosphorylated GSK-3ß level in the cortical neurons, which was prevented by both LY294002 and tricribine. CONCLUSION: These results suggest that activation of both the PI3K/Akt/mTOR and Akt/GSK-3ß signaling pathways is responsible for the atorvastatin-induced neurite outgrowth in cultured cortical neurons.

[Analyzing Japanese marsh warbler (Megalurus pryeri) song behavior in the breeding season].

The songs of Japanese marsh warblers (Megalurus pryeri) were recorded during May to July in 2009 at Shuangtaihekou Nature Reserve, Liaoning, China. Based on song characteristics, songs were divided into three types: courtship songs, alarm calls or contact calls. We analyzed and measured four parameters from 543 verses recorded from 20 males. The parameters were: duration of verse, number of syllables, duration of syllable, and interval of syllable. Verses of courtship song are formed of two verses, the first part's rhythm is more and more quick with time; the main body part is formed with complex syllables. Alarm calls and contact calls are simple, and formed with simple and repeat syllables. All songs contained 38 syllable types (six syllable types of the first part included). Acoustic features of the courtship song were statistically different, as was the calls of each individual.