GM3 synthase deficiency increases brain glucose metabolism in mice.

GM3 synthase (GM3S) deficiency is a rare neurodevelopmental disorder caused by an inability to synthesize gangliosides, for which there is currently no treatment. Gangliosides are brain-enriched, plasma membrane glycosphingolipids with poorly understood biological functions related to cell adhesion, growth, and receptor-mediated signal transduction. Here, we investigated the effects of GM3S deficiency on metabolism and mitochondrial function in a mouse model. By indirect calorimetry, GM3S knockout mice exhibited increased whole-body respiration and an increased reliance upon carbohydrate as an energy source. 18F-FDG PET confirmed higher brain glucose uptake in knockout mice, and GM3S deficient N41 neuronal cells showed higher glucose utilization in vitro. Brain mitochondria from knockout mice respired at a higher rate on Complex I substrates including pyruvate. This appeared to be due to higher expression of pyruvate dehydrogenase (PDH) and lower phosphorylation of PDH, which would favor pyruvate entry into the mitochondrial TCA cycle. Finally, it was observed that blocking glucose metabolism with the glycolysis inhibitor 2-deoxyglucose reduced seizure intensity in GM3S knockout mice following administration of kainate. In conclusion, GM3S deficiency may be associated with a hypermetabolic phenotype that could promote seizure activity.

Pregnancy loss and the risk of rheumatoid arthritis in Chinese women: findings from the China Kadoorie biobank.

Considering the female preponderance of rheumatoid arthritis (RA), and disease onset typically after the reproductive years, pregnancy and childbirth may play a role in the aetiology of the disease. Adverse outcomes of pregnancy have been found to precede the diagnosis of autoimmune diseases, including RA, but the evidence is scant and inconsistent. Therefore, we investigate whether pregnancy loss is associated with the risk of RA in Chinese women. Data from the China Kadoorie Biobank, conducted by the University of Oxford and the Chinese Centre for Disease Control and Prevention, of 299,629 Chinese women who had been pregnant were used. Multivariable logistic regression and stratified analyses were employed to analyse the association between types of pregnancy loss with the risk of RA. Pregnancy loss was significantly associated with increased risk of RA (OR 1.12, 95% CI 1.06-1.18), specifically, spontaneous (OR 1.11, 95% CI 1.03-1.20) and induced abortions (OR 1.11, 95% CI 1.06-1.17). There was no significant association between stillbirth and the risk of RA (OR 1.07, 95% CI 0.97-1.18). The risk of developing RA increases with the number of pregnancy losses: one loss confers an OR of 1.09 (95% CI 1.03-1.16), two an OR of 1.13 (95% CI 1.05-1.20), three or more an OR of 1.19 (95% CI 1.10-1.28) and OR of 1.06 (95% CI 1.03-1.08) for each additional. Spontaneous and induced abortions are associated with an increased risk of RA in Chinese women.

Octanoate is differentially metabolized in liver and muscle and fails to rescue cardiomyopathy in CPT2 deficiency.

Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart; thus, therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium-chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation-deficient hearts because of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate, but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability, whereas cardiac and skeletal muscles depend on carnitine but not on CPT2.

Role of mitochondrial acyl-CoA dehydrogenases in the metabolism of dicarboxylic fatty acids.

Dicarboxylic fatty acids, taken as a nutritional supplement or produced endogenously via omega oxidation of monocarboxylic fatty acids, may have therapeutic potential for rare inborn errors of metabolism as well as common metabolic diseases such as type 2 diabetes. Breakdown of dicarboxylic acids yields acetyl-CoA and succinyl-CoA as products, the latter of which is anaplerotic for the TCA cycle. However, little is known about the metabolic pathways responsible for degradation of dicarboxylic acids. Here, we demonstrated with whole-cell fatty acid oxidation assays that both mitochondria and peroxisomes contribute to dicarboxylic acid degradation. Several mitochondrial acyl-CoA dehydrogenases were tested for activity against dicarboxylyl-CoAs. Medium-chain acyl-CoA dehydrogenase (MCAD) exhibited activity with both six and 12 carbon dicarboxylyl-CoAs, and the capacity for dehydrogenation of these substrates was significantly reduced in MCAD knockout mouse liver. However, when dicarboxylic acids were fed to normal mice, the expression of MCAD did not change, while expression of peroxisomal fatty acid oxidation enzymes was greatly upregulated. In conclusion, mitochondrial fatty acid oxidation, and in particular MCAD, contributes to dicarboxylic acid degradation, but feeding dicarboxylic acids induces only the peroxisomal pathway.

The common K333Q polymorphism in long-chain acyl-CoA dehydrogenase (LCAD) reduces enzyme stability and function.

The fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed at high levels in human alveolar type II (ATII) cells in the lung. A common polymorphism causing an amino acid substitution (K333Q) was previously linked to a loss of LCAD antigen in the lung tissue in sudden infant death syndrome. However, the effects of the polymorphism on LCAD function has not been tested. The present work evaluated recombinant LCAD K333Q. Compared to wild-type LCAD protein, LCAD K333Q exhibited significantly reduced enzymatic activity. Molecular modeling suggested that K333 is within interacting distance of the essential FAD cofactor, and the K333Q protein showed a propensity to lose FAD. Exogenous FAD only partially rescued the activity of LCAD K333Q. LCAD K333Q protein was less stable than wild-type when incubated at physiological temperatures, likely explaining the observation of dramatically reduced LCAD antigen in primary ATII cells isolated from individuals homozygous for K333Q. Despite the effect of K333Q on activity, stability, and antigen levels, the frequency of the polymorphism was not increased among infants and children with lung disease.

Sirtuin 5 Regulates Proximal Tubule Fatty Acid Oxidation to Protect against AKI.

BACKGROUND: The primary site of damage during AKI, proximal tubular epithelial cells, are highly metabolically active, relying on fatty acids to meet their energy demands. These cells are rich in mitochondria and peroxisomes, the two organelles that mediate fatty acid oxidation. Emerging evidence shows that both fatty acid pathways are regulated by reversible posttranslational modifications, particularly by lysine acylation. Sirtuin 5 (Sirt5), which localizes to both mitochondria and peroxisomes, reverses post-translational lysine acylation on several enzymes involved in fatty acid oxidation. However, the role of the Sirt5 in regulating kidney energy metabolism has yet to be determined. METHODS: We subjected male Sirt5-deficient mice (either +/- or -/-) and wild-type controls, as well as isolated proximal tubule cells, to two different AKI models (ischemia-induced or cisplatin-induced AKI). We assessed kidney function and injury with standard techniques and measured fatty acid oxidation by the catabolism of 14C-labeled palmitate to 14CO2. RESULTS: Sirt5 was highly expressed in proximal tubular epithelial cells. At baseline, Sirt5 knockout (Sirt5-/- ) mice had modestly decreased mitochondrial function but significantly increased fatty acid oxidation, which was localized to the peroxisome. Although no overt kidney phenotype was observed in Sirt5-/- mice, Sirt5-/- mice had significantly improved kidney function and less tissue damage compared with controls after either ischemia-induced or cisplatin-induced AKI. This coincided with higher peroxisomal fatty acid oxidation compared with mitochondria fatty acid oxidation in the Sirt5-/- proximal tubular epithelial cells. CONCLUSIONS: Our findings indicate that Sirt5 regulates the balance of mitochondrial versus peroxisomal fatty acid oxidation in proximal tubular epithelial cells to protect against injury in AKI. This novel mechanism might be leveraged for developing AKI therapies.

An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein.

Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro. Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity. The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.

Upregulated IL-1 Receptor-associated Kinase 1 (IRAK1) in Systemic Lupus Erythematosus: IRAK1 Inhibition Represses Th17 Differentiation with Therapeutic Potential.

Systemic lupus erythematosus (SLE) is a typical autoimmune disease. Genome-wide analyses have revealed that interleukin-1 receptor-associated kinase 1 (IRAK1) is associated with susceptibility to SLE. Our previous study investigated the role of IRAK1 in nuclear factor-κB (NF-κB)-related pathways in a mouse model of lupus. In this study, we aimed to further explore the etiological role of IRAK1. The gene expression and phosphorylation of IRAK1 in CD4+ T cells from lupus patients and healthy controls were examined by quantitative reverse transcription-polymerase chain reaction and western blotting, respectively. The percentage of circulating Th17 cells and plasma IL-17A levels were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. The influence of IRAK1 suppression on Th17 development was assessed using an IRAK1 inhibitor and small interfering RNA. We found that IRAK1 transcript levels in CD4+ T cells were significantly upregulated in SLE patients in comparison to controls and were positively correlated with disease activity. In vitro experiments showed that lupus CD4+ T cells had more pronounced IRAK1 phosphorylation at threonine-209 upon IL-1ß stimulation than did control cells. Moreover, IRAK1 expression was positively associated with Th17/IL-17A in patients. When naïve CD4+ T cells were polarized toward the Th17 subset, IRAK1 inhibition significantly repressed IL-17A production and the gene expression of Th17 markers, namely, retinoic acid receptor-related orphan receptor c, IL-23 receptor and IL-17A. In summary, IRAK1 is overexpressed and hyperactivated in CD4+ T cells from SLE patients. IRAK1 inhibition attenuates Th17 differentiation in the context of human SLE, suggesting a therapeutic opportunity.

Curcumin prevents strokes in stroke-prone spontaneously hypertensive rats by improving vascular endothelial function.

BACKGROUND: Antioxidants have shown great promise in stroke prevention. Diarylheptanoids (also known as diphenylheptanoids) are a small class of plant secondary metabolites that possess antioxidant activity greater than that of α-tocopherol. Curcumin is the best known member and is mainly extracted from turmeric. This study aimed to explore whether curcumin has a preventive effect on stroke. METHODS: Stroke-prone spontaneously hypertensive rats (SHRsp) were randomly divided into control group (n = 10) and curcumin group (n = 10), and saline or curcumin (100 mg/kg/day) was administrated daily. Vascular endothelial function was examined by the relaxation of the artery in response to acetylcholine (ACH). The levels of reactive oxygen species (ROS) and nitric oxide (NO) were measured by using dihydroethidium (DHE) and 4, 5-diaminofluorescein (DAF-2 DA), respectively. The expression of uncoupling protein 2 (UCP2) was examined by RT-PCR and immunoblotting. RESULTS: Administration of curcumin significantly delayed the onset of stroke and increased the survival of SHRsp, which was ascribed to decreased ROS and improved endothelial dependent relaxation of carotid arteries. In the presence of UCP2 inhibitor genipin, both curcumin-mediated decrease of ROS and increase of NO production were blocked. CONCLUSION: Our study suggests that curcumin exerts a stroke preventive effect by attenuating oxidative stress to improve vascular endothelial function, which might be associated with UCP2 signaling.

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency.

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.

Aspirin increases mitochondrial fatty acid oxidation.

The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders.

Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site.

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.

Estimating low concentration heavy metals in water through hyperspectral analysis and genetic algorithm-partial least squares regression.

Hyperspectral spectrum enables assessment of heavy metal content, but research on low concentration in water is limited. This study employed in situ hyperspectral data from Dalian Lake, Shanghai to develop a machine learning model for accurately determining heavy metal concentrations. Initially, we employed a combination of empirical analysis and algorithm-based analysis to identify the optimal features for retrieving Cu and Fe ions. Based on the correlation coefficients between heavy metals and water quality, the feature bands for TOC, Chl-a and TP were selected as empirical features. Algorithm-based feature selection was conducted by employing the random forest (RF) approach with the original spectrum (OR), first-order derivative reflectance (FDR), and second-order derivative reflectance (SDR). For the development of a prediction model, we utilized the Genetic Algorithm-Partial Least Squares Regression (GA-PLSR) approach for Cu and Fe ions inversion. Our findings demonstrated that the integration of both empirical features and algorithm-selected features resulted in superior performance compared to using algorithm-selected features alone. Importantly, the crucial wavelength data primarily located at 497, 665, 686, 831 and 935 nm showed superior results for Cu retrieval, while wavelengths of 700, 746, 801, 948, and 993 nm demonstrated better results for Fe retrieval. These results also displayed that the GA-PLSR model outperformed both the PLSR and RF models, exhibiting an R2 of 0.75, RMSE of 0.004, and MRE of 0.382 for Cu inversion. For Fe inversion, the GA-PLSR model outperformed other models with an R2 of 0.73, RMSE of 0.036, and MRE of 0.464. This research provides a scientific basis and data support for monitoring low concentrations of heavy metals in water bodies using hyperspectral remote sensing techniques.

Sedentary Behavior and Physical Frailty Among Rural Older Adults in China: The Moderating Effect of Social Isolation.

OBJECTIVES: Few studies have explored the mechanisms underlying the relationship between sedentary behavior and physical frailty. The aim of this study was to investigate the moderating effect of social isolation on the association between sedentary behavior and physical frailty among older adults in rural China. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Data were from 3238 individuals aged ≥60 years from rural areas in China. METHODS: Binary logistic regression was used to explore the association between sedentary behavior and physical frailty and the moderating effect of social isolation. RESULTS: The prevalence of physical frailty was 18.7% among the older adults, and 17.0% of them were sedentary for ≥8 h/d. Compared with older adults with sedentary behavior for <4 h/d, participants with sedentary behavior for ≥8 h/d were more likely to suffer from physical frailty [odds ratio (OR), 2.26; 95% CI, 1.57-3.27]. We found that social isolation may aggravate this relationship (OR, 3.31; 95% CI, 2.06-5.32), especially for rural older adults who were sedentary for ≥8 h/day. CONCLUSION AND IMPLICATIONS: More sedentary behavior was associated with higher risk of physical frailty, which was especially apparent among older adults with social isolation, suggesting that sedentary older people who experienced social isolation were more vulnerable to physical frailty. Decreasing sedentary behavior in older adults and encouraging them to participate in interactive social activities could help prevent physical frailty.

Dietary dicarboxylic acids provide a non-storable alternative fat source that protects mice against obesity.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli.

G protein coupled receptors (GPCRs) are key players in signal recognition and cellular communication making them important therapeutic targets. Large-scale production of these membrane proteins in their native form is crucial for understanding their mechanism of action and target-based drug design. Here we report the overexpression system for a GPCR, the cannabinoid receptor subtype 2 (CB2), in Escherichia coli C43(DE3) facilitated by two fusion partners: Mistic, an integral membrane protein expression enhancer at the N-terminal, and TarCF, a C-terminal fragment of the bacterial chemosensory transducer Tar at the C-terminal of the CB2 open reading frame region. Multiple histidine tags were added on both ends of the fusion protein to facilitate purification. Using individual and combined fusion partners, we found that CB2 fusion protein expression was maximized only when both partners were used. Variable growth and induction conditions were conducted to determine and optimize protein expression. More importantly, this fusion protein Mistic-CB2-TarCF can localize into the E. coli membrane and exhibit functional binding activities with known CB2 ligands including CP55,940, WIN55,212-2 and SR144,528. These results indicate that this novel expression and purification system provides us with a promising strategy for the preparation of biologically active GPCRs, as well as general application for the preparation of membrane-bound proteins using the two new fusion partners described.

Reg1 protein regulates phosphorylation of all three Snf1 isoforms but preferentially associates with the Gal83 isoform.

The phosphorylation status of the Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation, catalyzed by the yeast PP1 phosphatase Glc7 in complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7, suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric ß subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association with the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore, nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex.

Manufacturing of Porous Glass by Femtosecond Laser Welding.

Based on femtosecond laser glass welding, four different porous structures of welding spots were formed by the manufacturing processes of spatiotemporal beam shaping and alternating high repetition rate transformation. Compared with an ordinary Gaussian beam, the welding spot fabricated by the flattened Gaussian beam had smoother welding edges with little debris, and the bottom of the welding spot pore was flat. Instead of a fixed high repetition rate, periodically alternating high repetition rates were adopted, which induced multiple refractive indices in the welding spot pore. The welding spot pores manufactured by spatiotemporal beam shaping and alternating high repetition rate transformation have a special structure and excellent properties, which correspond to superior functions of porous glass.

Association between pregnancy and pregnancy loss with COPD in Chinese women: The China Kadoorie Biobank study.

Background: Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease characterized by airflow blockage. Pregnancy and pregnancy loss may be related to an elevated risk of COPD, although studies have yet to report on this association. Hence, this study aims to investigate the association between pregnancy and pregnancy loss with the risk of COPD among Chinese women. Methods: Data on 302,510 female participants from the China Kadoorie Biobank were utilized for this study. Multivariable logistic regression, stratified by sociodemographic and lifestyle factors, was employed to obtain the odds ratio (ORs) and 95% confidence intervals (CIs) for the association between pregnancy and pregnancy loss with COPD. Results: Pregnancy loss was significantly associated with increased risk of COPD (OR 1.19, 95% CI 1.13-1.25), specifically, spontaneous (OR 1.19, 95% CI 1.11-1.29) and induced abortion (OR 1.18, 95% CI 1.12-1.25). Stillbirth, however, was not significantly associated with the risk of COPD (OR 1.09, 95% CI 0.99-1.20). Increasing number of pregnancy losses was associated with increasing risk of COPD (one pregnancy loss: OR 1.14, 95% CI 1.07-1.21, two or more pregnancy loss: OR 1.25, 95% CI 1.17-1.32, and each additional pregnancy loss: OR 1.06, 95% CI 1.03-1.09). A single pregnancy was significantly associated with reduced risk of COPD (OR 0.75, 95% CI 0.59-0.97), although each additional pregnancy was significantly associated with increased risk of COPD (OR 1.03, 95% CI 1.01-1.04). Conclusion: Pregnancy loss, in particular, spontaneous and induced abortions are associated with increased risk of COPD among Chinese women. A single pregnancy, however, demonstrated protective effects.