Organelle Formation in Bacteria and Archaea.

Uncovering the mechanisms that underlie the biogenesis and maintenance of eukaryotic organelles is a vibrant and essential area of biological research. In comparison, little attention has been paid to the process of compartmentalization in bacteria and archaea. This lack of attention is in part due to the common misconception that organelles are a unique evolutionary invention of the "complex" eukaryotic cell and are absent from the "primitive" bacterial and archaeal cells. Comparisons across the tree of life are further complicated by the nebulous criteria used to designate subcellular structures as organelles. Here, with the aid of a unified definition of a membrane-bounded organelle, we present some of the recent findings in the study of lipid-bounded organelles in bacteria and archaea.

A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum.

Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.

Intrinsic and extrinsic determinants of conditional localization of Mms6 to magnetosome organelles in Magnetospirillum magneticum AMB-1.

Magnetotactic bacteria are a diverse group of microbes that use magnetic particles housed within intracellular lipid-bounded magnetosome organelles to guide navigation along geomagnetic fields. The development of magnetosomes and their magnetic crystals in Magnetospirillum magneticum AMB-1 requires the coordinated action of numerous proteins. Most proteins are thought to localize to magnetosomes during the initial stages of organelle biogenesis, regardless of environmental conditions. However, the magnetite-shaping protein Mms6 is only found in magnetosomes that contain magnetic particles, suggesting that it might conditionally localize after the formation of magnetosome membranes. The mechanisms for this unusual mode of localization to magnetosomes are unclear. Here, using pulse-chase labeling, we show that Mms6 translated under non-biomineralization conditions translocates to pre-formed magnetosomes when cells are shifted to biomineralizing conditions. Genes essential for magnetite production, namely mamE, mamM, and mamO, are necessary for Mms6 localization, whereas mamN inhibits Mms6 localization. MamD localization was also investigated and found to be controlled by similar cellular factors. The membrane localization of Mms6 is dependent on a glycine-leucine repeat region, while the N-terminal domain of Mms6 is necessary for retention in the cytosol and impacts conditional localization to magnetosomes. The N-terminal domain is also sufficient to impart conditional magnetosome localization to MmsF, altering its native constitutive magnetosome localization. Our work illuminates an alternative mode of protein localization to magnetosomes in which Mms6 and MamD are excluded from magnetosomes by MamN until biomineralization initiates, whereupon they translocate into magnetosome membranes to control the development of growing magnetite crystals.IMPORTANCEMagnetotactic bacteria (MTB) are a diverse group of bacteria that form magnetic nanoparticles surrounded by membranous organelles. MTB are widespread and serve as a model for bacterial organelle formation and biomineralization. Magnetosomes require a specific cohort of proteins to enable magnetite formation, but how those proteins are localized to magnetosome membranes is unclear. Here, we investigate protein localization using pulse-chase microscopy and find a system of protein coordination dependent on biomineralization-permissible conditions. In addition, our findings highlight a protein domain that alters the localization behavior of magnetosome proteins. Utilization of this protein domain may provide a synthetic route for conditional functionalization of magnetosomes for biotechnological applications.

Chemodivergence in Fluorine Source-Controlled Cascade Reaction of Aryne Precursors to Synthesize Pyrrolo[3,4-b]indoles and 3-Arylated Maleimides.

Reactions allowing chemodivergence prove to be attractive strategies in synthetic organic chemistry. We herein described a highly practical, transition-metal-free, highly regioselective and chemodivergent cascade reaction controlled by fluorine sources, which involved a [3 + 2] cycloaddition or C-arylation process between aryne precursors and 3-aminomaleimides. These two pathways led to a wide scope of structurally diverse pyrrolo[3,4-b]indoles (19 examples) and 3-arylated maleimides (25 examples) in good-to-excellent yields. Furthermore, the reaction could be scaled up, and several synthetic transformations were accomplished for the preparation of functionalized molecules and might provide new opportunities for the discovery of N-heterocyclic drugs.

Base-Catalyzed Cascade Cyclization of 2-Nitrochalcones and Isocyanides to Access Pyrano[3,4-b]indol-1(9H)-one Frameworks.

An unexpected cascade reaction of 2-nitrochalcones with isocyanoacetates has been reported for the efficient synthesis of indole carboxylic esters and pyranoindoles. The conversion was achieved by KOH-catalyzed cyclization and elimination of the nitro group with final decarbonylation-aromatization. The method was used to synthesize a series of potentially biologically active indole derivatives (49 examples) in 67-85% yields under transition-metal-free catalytic conditions.

Association between transition patterns of sleep problems and suicidal ideation in Chinese female nurses: A prospective study.

OBJECTIVE: Suicidal ideation and sleep problems are both common in nurses. However, few longitudinal studies are available to examine the temporal association between sleep and suicidal ideation in nurses. METHOD: Data from the Health Longitudinal Survey of Nurses in Shandong Province was analyzed, involving 623 female nurses who had completed data of concern in 2018 (T1) and 2019 (T2). Sleep problem was assessed by the Pittsburgh Sleep Quality Index, in which the transition patterns for global and specific sleep component and the cumulative number of sleep component problems were defined. Suicidal ideation was measured by the ninth item of the Patient Health Questionnaire. Binary logistic regression was used to explore the association between sleep and suicidal ideation. RESULTS: Chronic and deteriorated global sleep problems is associated with a greater risk of suicidal ideation. For the specific component of sleep, sleep disturbance and short sleep duration are associated with a higher risk of suicidal ideation. The higher number of cumulative sleep component problems is associated with a higher risk of suicidal ideation. CONCLUSION: Findings indicate sleep disturbance and short sleep duration may be pathways to suicidal ideation. Initiatives that target at sleep problems may be important to reduce suicidal ideation in nurses.

Melanoma differentiation-Associated gene 5 protects against NASH in mice.

BACKGROUND AND AIMS: NASH is a complicated disease characterized by hepatocyte steatosis, inflammation infiltration, and liver fibrosis. Accumulating evidence suggests that the innate immunity plays a key role in NASH progression. Here, we aimed to reveal the role of melanoma differentiation-associated gene 5 (MDA5, also known as Ifih1), a conventional innate immune regulator following viral infection, in the progression of NASH and investigate its underlying mechanism. APPROACH AND RESULTS: We first examined the expression of MDA5 and found that MDA5 was markedly down-regulated in the livers with NASH in human individuals and mice models. MDA5 overexpression significantly inhibits the free fatty acid-induced lipid accumulation and inflammation in hepatocyte in vitro, whereas MDA5 knockdown promotes hepatocyte lipotoxicity. Using hepatocyte-specific Mda5 gene knockout and transgenic mice, we found that diet-induced hepatic steatosis, inflammation, and liver fibrosis were markedly exacerbated by Mda5 deficiency but suppressed by Mda5 overexpression. Mechanistically, we found that the activation of apoptosis signal-regulating kinase 1 (ASK1)-mitogen-activated protein kinase pathway was significantly inhibited by MDA5 but enhanced by MDA5 deletion. We further validated that MDA5 directly interacted with ASK1 and suppressed its N-terminal dimerization. Importantly, blockage of ASK1 with adenovirus-expressing dominant negative ASK1 obviously reversed the lipid accumulation and ASK1 pathway activation when Mda5 was knocked out. CONCLUSIONS: These data indicate that MDA5 is an essential suppressor in NASH. The findings support MDA5 as a regulator of ASK1 and a promising therapeutic target for NASH.

Cu(II)-Catalyzed Cascade of N-Phenyl-o-phenylenediamine with Benzaldehyde: One-Step Direct Construction of 2-(1-Phenyl-1H-benzo[d]imidazol-2-yl)phenols.

A practical protocol for the construction of hydroxylated 2-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenols (PBIs) from N-phenyl-o-phenylenediamine with benzaldehydes was developed. The cascade reaction was enabled by heating a mixture of the two substrates in the presence of air as an oxidant and anhydrous Cu(OAc)2 as a catalyst in dimethyl sulfoxide, and a diverse series of PBIs were synthesized in moderate to good yields (69-81%). Furthermore, the synthesis of the PBIs was enabled via a one-pot cascade reaction that proceeded through subsequent dehydration condensation, intramolecular cyclization, and aromatic C-H hydroxylation. This protocol can be used for the synthesis of hydroxylated PBI via a one-pot annulation C-H hydroxylation reaction rather than through a series of multistep reactions, which provides the possibility of further modification.

Gastrodin Improves Nonalcoholic Fatty Liver Disease Through Activation of the Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

BACKGROUND AND AIMS: NASH is currently one of the most common causes of liver transplantation and hepatocellular carcinoma. Thus far, there is still no effective pharmacological therapy for this disease. Recently, Gastrodin has demonstrated hepatoprotective effects in a variety of liver diseases. The aim of this study is to investigate the function of Gastrodin in NASH. APPROACH AND RESULTS: In our study, Gastrodin showed potent therapeutic effects on NASH both in vivo and in vitro. In high-fat diet or high-fat and high-cholesterol diet-fed mice, the liver weight, hepatic and serum triglyceride and cholesterol contents, and serum alanine aminotransferase and aspartate aminotransferase activity levels were markedly reduced by Gastrodin treatment as compared with the corresponding vehicle groups. Notably, Gastrodin showed minimal effects on the function and histological characteristics of other major organs in mice. We further examined the effects of Gastrodin on lipid accumulation in primary mouse hepatocytes and human hepatocyte cell line and observed that Gastrodin showed a significant decrease in lipid accumulation and inflammatory response in hepatocytes under metabolic stress. Furthermore, RNA-sequencing analysis systemically indicated that Gastrodin suppressed the pathway and key regulators related to lipid accumulation, inflammation, and fibrosis in the pathogenesis of NASH. Mechanistically, we found that Gastrodin protected against NASH by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway, which was supported by the result that the AMPK inhibitor Compound C or AMPK knockdown blocked the Gastrodin-mediated hepatoprotective effect. CONCLUSIONS: Gastrodin attenuates steatohepatitis by activating the AMPK pathway and represents a therapeutic for the treatment of NASH.

SIMPLE Is an Endosomal Regulator That Protects Against NAFLD by Targeting the Lysosomal Degradation of EGFR.

BACKGROUND AND AIMS: NAFLD has become a tremendous burden for public health; however, there is no drug for NAFLD therapy at present. Impaired endo-lysosome-mediated protein degradation is observed in a variety of metabolic disorders, such as atherosclerosis, type 2 diabetes mellitus, and NAFLD. Small integral membrane protein of lysosome/late endosome (SIMPLE) is a regulator of endosome-to-lysosome trafficking and cell signaling, but the role that SIMPLE plays in NAFLD progression remains unknown. Here we investigated SIMPLE function in NAFLD development and sophisticated mechanism therein. APPROACH AND RESULTS: This study found that in vitro knockdown of SIMPLE significantly aggravated lipid accumulation and inflammation in hepatocytes treated with metabolic stimulation. Consistently, in vivo experiments showed that liver-specific Simple-knockout (Simple-HKO) mice exhibited more severe high-fat diet (HFD)-induced, high-fat-high-cholesterol diet (HFHC)-induced, and methionine-choline-deficient diet (MCD)-induced steatosis, glucose intolerance, inflammation, and fibrosis than those fed with normal chow (NC) diet. Meanwhile, RNA-sequencing demonstrated the up-regulated signaling pathways and signature genes involved in lipid metabolism, inflammation, and fibrosis in Simple-HKO mice compared with control mice under metabolic stress. Mechanically, we found SIMPLE directly interact with epidermal growth factor receptor (EGFR). SIMPLE deficiency results in dysregulated degradation of EGFR, subsequently hyperactivated EGFR phosphorylation, thus exaggerating NAFLD development. Moreover, we demonstrated that using EGFR inhibitor or silencing EGFR expression could ameliorate lipid accumulation induced by the knockdown of SIMPLE. CONCLUSIONS: SIMPLE ameliorated NASH by prompting EGFR degradation and can be a potential therapeutic candidate for NASH.

Exosomes from M2-polarized macrophages relieve oxygen/glucose deprivation/normalization-induced neuronal injury by activating the Nrf2/HO-1 signaling.

Stroke is a life-threatening neurological disorder with limited therapeutic efficacy. Previous studies have demonstrated that macrophages play an important role in brain injury after a stroke. However, its underlying mechanism remains unclear and the role of exosomes derived from M2-polarized macrophages (M2-Exo) in ischemic stroke has not yet been reported. In this study, we established an in vitro oxygen/glucose deprivation and re-oxygen/glucose (OGD/R) model to investigate the potential role of M2-Exo in protecting HT22 neurons against ischemia-reperfusion injury. Interleukin-4 was used to induce the M2 phenotype in macrophages, following which the exosomes were isolated from the supernatant of M2-polarized macrophages and identified by western blotting, transmission electron microscopy, and nanoparticle tracking analysis. After co-incubation with M2-Exo, OGD/R-induced neuronal injury in HT22 cells was improved, accompanied by increased cell viability and decreased lactate dehydrogenase release. In addition, the increase in percentage of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling-positive cells in OGD/R-treated HT22 cells was attenuated after incubation with M2-Exo. M2-Exo treatment also suppressed reactive oxygen species and malondialdehyde production and improved the reduction of superoxide dismutase activity. Moreover, M2-Exo treatment was found to activate the nuclear factor erythroid related factor 2 (Nrf2)/heme-oxygenase-1 (HO-1) signaling pathway in OGD/R-treated HT22 neurons. Importantly, inhibition of Nrf2 by ML385 partially reversed the protective effects of M2-Exo against OGD/R-induced oxidative damage. Taken together, these data demonstrated that M2-Exo exerted protective effects against OGD/R-induced oxidative damage in HT22 neurons, which was mediated by the activation of Nrf2/HO-1 signaling. Hence, our findings provide a promising therapeutic approach for ischemic stroke.

MiR-1224 downregulation inhibits OGD/R-induced hippocampal neuron apoptosis through targeting Ku protein.

Ischemic cerebrovascular disease is the main cause of disability due to stroke. This study aimed to investigate the function of miR-1224 in OGD/R-induced hippocampal neuron apoptosis, as well as the regulatory mechanism of miR-1224 in ischemic cerebrovascular disease. The oxygen-glucose deprivation/reperfusion (OGD/R) model of primary mouse hippocampal neurons was established. RT-qPCR detected miR-1224, Ku70 and Ku86 levels. Western blotting was applied to measure the expression of Ku70/86 and apoptosis related proteins. Flow cytometry was used to assess apoptosis. JC-1 fluorescence was performed to test the mitochondrial membrane potential (MMP) in neurons. The double luciferase reporter assay was performed to investigate the relationship between miR-1224 and Ku70/86. OGD/R induced the apoptosis and mitochondrial injury in neuronal cells, while miR-1224 downregulation or Ku70/86 upregulation reversed this phenomenon. Meanwhile, miR-1224 negatively regulated the expression of Ku70/86 in neuronal cells through directly targeting Ku70/86. Furthermore, knockdown of Ku70/86 significantly reversed the inhibitory effect of miR-1224 silencing on apoptosis and mitochondrial injury in OGD/R-treated neuronal cells. Our findings indicated that miR-1224 downregulation suppressed OGD/R-induced hippocampal neuron apoptosis by targeting Ku protein, suggesting that miR-1224 could serve as a new target for ischemic cerebrovascular disease treatment.

Tripartite-motif protein 21 knockdown extenuates LPS-triggered neurotoxicity by inhibiting microglial M1 polarization via suppressing NF-κB-mediated NLRP3 inflammasome activation.

Tripartite motif-containing 21 (TRIM21) has been confirmed to mediate the production of inflammatory mediators via NF-κB signaling. However, the function of TRIM21 in microglia-mediated neuroinflammation remains unclear. This study aimed to explore the effect of TRIM21 on LPS-activated BV2 microglia and its underlying mechanism. BV2 cells exposed to lipopolysaccharide (LPS) were used to simulated neuroinflammation in vitro. Loss-of-function and gain-of-function of TRIM21 in BV2 cells were used to assess the effect of TRIM21 on LPS-induced neuroinflammation. BV2 microglia and HT22 cells co-culture system were used to investigate whether TRIM21 regulated neuronal inflammation-mediated neuronal death. TRIM21 knockdown triggered the polarization of BV2 cells from M1 to M2 phenotype. Knockdown of TRIM21 reduced the secretion of TNF-α, IL-6, and IL-1ß, while increased the content of IL-4 in LPS-treated cells. Knockdown of TRIM21 inhibited the expression of p65 and the binding activity of NF-κB-DNA. Additionally, TRIM21 siRNA eliminated the increase in NLRP3 and cleaved caspase-1 proteins expression and caspase-1 activity induced by LPS. TRIM21 knockdown could resist cytotoxicity induced by activated microglia, including increasing the viability of co-cultured HT22 cells and reducing the emancipation of LDH. Moreover, the increased apoptosis and caspase-3 activity of HT22 neurons induced by activated BV2 cells were blocked by TRIM21 siRNA. Blocking of NF-κB abolished the effect of TRIM21 in promoting the expression of M1 phenotype marker genes. Similarly, the blockade of NF-κB pathway eliminated the promotion of TRIM21 on neurotoxicity induced by neuroinflammation. TRIM21 knockdown suppressed the M1 phenotype polarization of microglia and neuroinflammation-mediated neuronal damage via NF-κB/NLRP3 inflammasome pathway, which suggested that TRIM21 might be a potential therapeutic target for the therapy of central nervous system diseases.

Ketogenic diet and growth in Chinese infants with refractory epilepsy.

BACKGROUND AND OBJECTIVES: This study evaluated the impact of 12 months of ketogenic dietary treatment (KDT) on growth in Chinese infants with refractory epilepsy. METHODS AND STUDY DESIGN: The KDT group included patients who were divided into groups A (age 6-12 months), B (12-24 months) and C (24-36 months). The normal group included infants aged approximately 6-12 months, 12-24 months and 24-36 months who were classified into groups A1, B1 and C1, respectively. Data on height, weight, aspartate transaminase (AST), alanine transaminase (ALT), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TGs), zinc, iron, calcium, magnesium, and haemoglobin (Hb) were extracted from the medical records. Then, we compared the impacts of 12 months of KDT on growth. RESULTS: Forty-one patients were included in the KDT group, and 90 infants were included in the normal group. The overall prevalence of underweight (WAZ <-2 SD), stunting (HAZ <-2 SD), wasting (BAZ <-2 SD), and overweight/obesity (BAZ ≥2 SD) were relatively lower in the A and B groups. The prevalence of anaemia in group A was significantly higher than that in group A1. No significant differences were observed in the KDT groups with regard to HDL, LDL, AST, ALT, iron, calcium, magnesium, or zinc. A greater than 50% reduction in weekly seizure frequency was evident in 100% of group A, 78.6% of group B and 77.8% of group C. CONCLUSIONS: The results revealed that patients less than 2 years old who received KDT maintained appropriate growth at the 12-month follow-up.

Magnetotactic Bacteria Accumulate a Large Pool of Iron Distinct from Their Magnetite Crystals.

Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that form intracellular nanoparticles of magnetite (Fe3O4) or greigite (Fe3S4) in a genetically controlled manner. Magnetite and greigite synthesis requires MTB to transport a large amount of iron from the environment. Most intracellular iron was proposed to be contained within the crystals. However, recent mass spectrometry studies suggest that MTB may contain a large amount of iron that is not precipitated in crystals. Here, we attempted to resolve these discrepancies by performing chemical and magnetic assays to quantify the different iron pools in the magnetite-forming strain Magnetospirillum magneticum AMB-1, as well as in mutant strains showing defects in crystal precipitation, cultivated at various iron concentrations. All results show that magnetite represents at most 30% of the total intracellular iron under our experimental conditions and even less in the mutant strains. We further examined the iron speciation and subcellular localization in AMB-1 using the fluorescent indicator FIP-1, which was designed for the detection of labile Fe(II). Staining with this probe suggests that unmineralized reduced iron is found in the cytoplasm and associated with magnetosomes. Our results demonstrate that, under our experimental conditions, AMB-1 is able to accumulate a large pool of iron distinct from magnetite. Finally, we discuss the biochemical and geochemical implications of these results.IMPORTANCE Magnetotactic bacteria (MTB) produce iron-based intracellular magnetic crystals. They represent a model system for studying iron homeostasis and biomineralization in microorganisms. MTB sequester a large amount of iron in their crystals and have thus been proposed to significantly impact the iron biogeochemical cycle. Several studies proposed that MTB could also accumulate iron in a reservoir distinct from their crystals. Here, we present a chemical and magnetic methodology for quantifying the iron pools in the magnetotactic strain AMB-1. Results showed that most iron is not contained in crystals. We then adapted protocols for the fluorescent Fe(II) detection in bacteria and showed that iron could be detected outside crystals using fluorescence assays. This work suggests a more complex picture for iron homeostasis in MTB than previously thought. Because iron speciation controls its fate in the environment, our results also provide important insights into the geochemical impact of MTB.

Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves.

Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis (Arabidopsis thaliana). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection.

Synaptotagmin 1 Is Involved in Neuropathic Pain and Electroacupuncture-Mediated Analgesic Effect.

Numerous studies have verified that electroacupuncture (EA) can relieve neuropathic pain through a variety of mechanisms. Synaptotagmin 1 (Syt-1), a synaptic vesicle protein for regulating exocytosis of neurotransmitters, was found to be affected by EA stimulation. However, the roles of Syt-1 in neuropathic pain and EA-induced analgesic effect remain unclear. Here, the effect of Syt-1 on nociception was assessed through an antibody blockade, siRNA silencing, and lentivirus-mediated overexpression of spinal Syt-1 in rats with spared nerve injury (SNI). EA was used for stimulating bilateral "Sanjinjiao" and "Zusanli" acupoints of the SNI rats to evaluate its effect on nociceptive thresholds and spinal Syt-1 expression. The mechanically and thermally nociceptive behaviors were assessed with paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different temperatures, respectively, at day 0, 7, 8, 14, and 20. Syt-1 mRNA and protein levels were determined with qRT-PCR and Western blot, respectively, and its distribution was observed with the immunohistochemistry method. The results demonstrated Syt-1 antibody blockade and siRNA silencing increased ipsilateral PWTs and PWLs of SNI rats, while Syt-1 overexpression decreased ipsilateral PWTs and PWLs of rats. EA significantly attenuated nociceptive behaviors and down-regulated spinal Syt-1 protein levels (especially in laminae I-II), which were reversed by Syt-1 overexpression. Our findings firstly indicate that Syt-1 is involved in the development of neuropathic pain and that EA attenuates neuropathic pain, probably through suppressing Syt-1 protein expression in the spinal cord.

The effect of temporal concept on the automatic activation of spatial representation: From axis to plane.

Temporal concepts could be represented horizontally(X-axis) or vertically (Y-axis). However, whether the spatial representation of time exists in the whole plane remains unclear. In this study, we investigated whether processing temporal concepts would automatically activate spatial representations in a whole plane without any guidance or cue. Participants first indicated whether a word was past-related or future-related, then, they identified a target in different visual fields. In Experiment 1, the results demonstrated that past time mapped onto the left and top in a plane or axis, while future time mapped onto the right and bottom, with the horizontal effect being stronger than the vertical effect. In Experiment 2, an index of eye movement showed a similar data pattern. Thinking about temporal concepts activates spatial schema automatically without guidance or cue, and the time-space metaphor is represented not only as an axis but also as a whole plane. The results were discussed in terms of the possible cultural differences that made the Chinese participants tend to be more flexible in spatial representation of time because of their comprehensive thinking.

Akt-mTOR Signaling Mediates Abnormalities in the Proliferation and Apoptosis of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome.

BACKGROUND/AIMS: Abnormal apoptosis of granulosa cells (GCs) is thought to involve in the pathogenesis of polycystic ovary syndrome (PCOS); however, the associated cellular and molecular mechanisms remain unclear. METHODS: Primary GCs were obtained from healthy women and women with PCOS. The cell proliferation and apoptosis were analyzed in insulin-stimulated and insulin receptor gene (INSR) siRNA-transfected GCs. The protein expression of Akt-mTOR-S6K1 signal molecules was measured by Western blot. RESULTS: This study showed that 1 nM of insulin significantly stimulated cell proliferation, induced cell apoptosis, and decreased the telomerase activity in GCs from both the healthy women and PCOS patients (p < 0.001), but silencing of INSR expression blocked the effects of insulin. Insulin induced significantly more apoptosis in GCs from PCOS patients than from healthy women (p < 0.01). Insulin significantly increased the ratio of p-Akt/Akt, the expression of mTOR protein, and the ratio of p-S6K1/S6K1 in GCs from normal control than in cells from PCOS patients (p < 0.001). CONCLUSION: Insulin-induced apoptosis of GCs, less activation of Akt-mTOR signaling, and reduction of telomerase activity may be associated with the pathogenesis of PCOS.

Decolorization of textile dye RB19 using volcanic rock matrix immobilized Bacillus thuringiensis cells with surface displayed laccase.

A triplicate volcanic rock matrix-Bacillus thuringiensis-laccase WlacD (VRMs-Bt-WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)2 as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)2-WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L 9(34)-orthogonal test, Plackett-Burman test, steepest ascent method, and Box-Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L-1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs-Bt-WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs-Bt-WlacD toward an initial concentration of 500 mg L-1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g-100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs-Bt-WlacD and have the potential for large-scale or continuous operations.