Micro-environment-triggered chemodynamic treatment for boosting bacteria elimination at low-temperature by synergistic effect of photothermal treatment and nanozyme catalysis.

An injectable hydrogel dressing, Zr/Fc-MOF@CuO2@FH, was constructed by combing acid-triggered chemodynamic treatment (CDT) with low-temperature photothermal treatment (LT-PTT) to effectively eliminate bacteria without harming the surrounding normal tissues. The Zr/Fc-MOF acts as both photothermal reagent and nanozyme to generate reactive oxygen species (ROS). The CuO2 nanolayer can be decomposed by the acidic microenvironment of the bacterial infection to release Cu2+ and H2O2, which not only induces Fenton-like reaction but also enhances the catalytic capability of the Zr/Fc-MOF. The generated heat augments ROS production, resulting in highly efficient bacterial elimination at low temperature. Precisely, injectable hydrogel dressing can match irregular wound sites, which shortens the distance of heat dissipation and ROS diffusion to bacteria, thus improving sterilization efficacy and decreasing non-specific systemic toxicity. Both in vitro and in vivo experiments validated the predominant sterilization efficiency of drug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and kanamycin-resistant Escherichia coli (KREC), presenting great potential for application in clinical therapy.

m6Am methyltransferase PCIF1 negatively regulates ciliation by inhibiting BICD2 expression.

N6, 2'-O-dimethyladenosine (m6Am) is a widespread RNA modification catalyzed by the methyltransferase PCIF1 (phosphorylated CTD interacting factor 1). Despite its prevalence, the biological functions of m6Am in RNA remain largely elusive. Here, we report a critical role of PCIF1-dependent m6Am RNA modification in ciliogenesis in RPE-1 cells. Our findings demonstrate that PCIF1 acts as a negative regulator of ciliation through its m6Am methyltransferase activity. A quantitative proteomic analysis identifies BICD2 as a downstream target of PCIF1, with PCIF1 depletion resulting in a significant increase in BICD2 levels. BICD2 depletion leads to a significant reduction in ciliation. Crucially, the ciliary phenotype in PCIF1-depleted cells is reversed upon BICD2 knockdown. Further investigations reveal that PCIF1 regulates BICD2 protein levels through its m6Am catalytic activity, which reduces the stability and translation efficiency of BICD2 mRNA. Single-base resolution LC-MS analysis identifies the m6Am site on BICD2 mRNA modified by PCIF1. These findings establish the essential involvement of PCIF1-dependent m6Am modification in ciliogenesis.

Reusable and self-sterilization mask for real-time personal protection based on sunlight-driven photocatalytic reaction.

Personal protective masks play critical role in preventing the disease epidemic and resisting pathogenic bacterial infestation. However, large quantities of masks were disposed during COVID-19 epidemic, which caused environmental problem and huge economic burden. Herein, we developed reusable masks with inherent antimicrobial and self-cleaning features under solar irradiation. With spun-bonded nonwoven fabrics (SNF) layer as substrate, copper sulfide@polydopamine nanoparticles are deposited on SNF layer (CuS@PDANPs-SNF), which presents excellent photocatalytic activity. Under solar irradiation, CuS@PDANPs produce abundant of singly linear oxygen (1O2), which inactivates pathogenic bacteria with high efficiency over 99%. Interestingly, CuS@PDANPs-SNF cannot cause high temperature to bring any uncomfortable to the person, which is suitable for human to wear in daily life. Such design effectively protect person from the transmission of viral aerosol. Meanwhile, CuS@PDANPs-SNF masks are reusable and still maintain robust bactericidal ability after washing. The sunlight-mediated self-sterilization at low temperature endows CuS@PDANPs-SNF masks as powerful personal protective equipment for daily protection, which also provides an instructive way for reducing the environmental impact.

Glucose-metabolism-triggered colorimetric sensor array for point-of-care differentiation and antibiotic susceptibility testing of bacteria.

Simple and sensitive discrimination of multiple bacteria and antimicrobial susceptibility test (AST) are significant for food safety, clinical diagnosis and treatment. Herein, based on different metabolic ability of bacteria on glucose, we presented a colorimetric sensor array for point-of-care testing (POCT) of multiple bacteria with methyl red (MER), bromothymol blue (BTB) and bromocresol green (BCG) as probes. Different bacteria resulted in different color changes of three probes, which was converted to RGB (Red (R)/Green (G)/Blue (B)) signals by the color recognizer APP loaded on smartphone. The sensor array performed differentiation of eleven species of bacteria, achieving the quantitative analysis of individual bacteria in tap water and differentiation of bacterial mixtures. Interestingly, the sensor array can be used for AST and evaluating minimal inhibitory concentration (MIC) of antibiotics to bacteria. The research provided meaningful guidance for distinguishing multiple bacteria and evaluating MIC, presenting great potential in practical application.

AIE Nanozyme-Based Long Persistent Chemiluminescence and Fluorescence for POCT of Pathogenic Bacteria.

Pathogenic bacteria are widely distributed in diverse environments and significantly threaten human health. Point-of-care testing (POCT) is a valuable way for early warnings of bacteria threat. Herein, a chemiluminescence (CL)-based ratiometric sensing platform was constructed for sensitive POCT of bacteria according to a newly designed aggregation-induced emission (AIE) molecule. The new AIE molecule presents oxidase-like properties (named as AIEzyme) and can trigger long persistent CL of luminol (LUM) with strong intensity in the absence of H2O2. The CL emission can be monitored with the naked eye for over 2 h. The emission mechanism is explored and may be attributed to the persistent reactive oxygen species generation of the AIEzyme according to the cyclic energy transfer between the AIEzyme and luminol, which catalyzes CL of luminol. Based on the CL resonance energy transfer mechanism, an afterglow luminescence system is further developed, which is used to construct a ratiometric biosensor for detection of pathogenic bacteria. With a homemade holder as a detection room and a smartphone as an analyzer, the portable biosensing platform is used for quantitative POCT of bacteria in real samples with good recovery. The detection is free of H2O2 and an external excitation source, which not only simplifies the operation but reduces interference. Specifically, the long persistent luminescence and the ratiometric strategy can significantly improve accuracy, providing an instructive way for point-of-need analysis, for example, SARS-CoV-2 detection and bioimaging analysis.

AIE Bioconjugates for Accurate Identification and In Vivo Targeted Treatment of Bacterial Infection Based on Bioorthogonal Reaction.

Targeted killing multidrug-resistant bacteria with high efficiency is urgently needed for the treatment of infection with minimal collateral damage. Herein, a new near-infrared (NIR) fluorescence nanoprobe is designed and synthesized with aggregation-induced emission (AIE) features, which also is excellent reactive oxygen species (ROS) generator. The as-prepared AIE nanoparticles (NPs) present outstanding sterilizing rate on methicillin-resistant Staphylococcus aureus (MRSA) and kanamycin-resistant Escherichia coli (KREC). Meanwhile, considering the differences in the surface structure of animal cells and bacteria, a non-invasive image-guided strategy for precise treatment of bacterial infection has been successfully implemented based on bioorthogonal reaction which can perform and control unnatural chemical reactions inside living organisms. The AIE NPs are thus specifically trapped on the bacterial surface while not on the normal cells, realizing real-time tracking of the infected site distribution in vivo and guiding photodynamic therapy (PDT) for eliminating bacteria in inflammation region. That significantly improves the accuracy and sterilization rate of bacterial-infected wounds with negligible side effects. The investigation developed a potential antibacterial agent and also provides an instructive way for targeting treatment based on bioorthogonal reaction.

Alteration of Chain-Length Selectivity and Thermostability of Rhizopus oryzae Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design.

Rhizopus oryzae lipase (ROL) is one of the most important enzymes used in the food, biofuel, and pharmaceutical industries. However, the highly demanding conditions of industrial processes can reduce its stability and activity. To seek a feasible method to improve both the catalytic activity and the thermostability of this lipase, first, the structure of ROL was divided into catalytic and noncatalytic regions by identifying critical amino acids in the crevice-like binding pocket. Second, a mutant screening library aimed at improvement of ROL catalytic performance by virtual saturation mutagenesis of residues in the catalytic region was constructed based on Rosetta's Cartesian_ddg protocol. A double mutant, E265V/S267W (with an E-to-V change at residue 265 and an S-to-W change at residue 267), with markedly improved catalytic activity toward diverse chain-length fatty acid esters was identified. Then, computational design of disulfide bonds was conducted for the noncatalytic amino acids of E265V/S267W, and two potential disulfide bonds, S61C-S115C and E190C-E238C, were identified as candidates. Experimental data validated that the variant E265V/S267W/S61C-S115C/E190C-E238C had superior stability, with an increase of 8.5°C in the melting temperature and a half-life of 31.7 min at 60°C, 4.2-fold longer than that of the wild-type enzyme. Moreover, the variant improved the lipase activity toward five 4-nitrophenyl esters by 1.5 to 3.8 times, exhibiting a potential to modify the catalytic efficiency. IMPORTANCE Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.

Deciphering cilia and ciliopathies using proteomic approaches.

Cilia are microtubule-based organelles that protrude from the cell surface and play crucial roles in cellular signaling pathways and extracellular fluid movement. Defects in the ciliary structures and functions are implicated in a set of hereditary disorders, including polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome, which are collectively termed as ciliopathies. The application of mass spectrometry-based proteomic approaches to explore ciliary components provides important clues for understanding their physiological and pathological roles. In this review, we focus primarily on proteomic studies involving the identification of proteins in motile cilia and primary cilia, proteomes in ciliopathies, and interactomes of ciliopathy proteins. Collectively, the integration of these data sets will be beneficial for the comprehensive understanding of ciliary structures and exploring potential biomarkers and therapeutic targets for ciliopathies.

Activated autophagy-lysosomal pathway in dairy cows with hyperketonemia is associated with lipolysis of adipose tissues.

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood ß-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 µg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of ß-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of ß-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.

Long-Lasting Chemiluminescence-Based POCT for Portable and Visual Pathogenic Detection and In Situ Inactivation.

Bacterial infections seriously threaten human health and also bring huge financial burden. It is critical to construct multifunctional platforms for effectively inactivating bacteria right after point-of-care testing (POCT). Chemiluminescence (CL) bioassays are considered as powerful candidates for POCT as they are free from using an excitation light source, while the flash-type emission limits their further application. Herein, a CL system with long, persistent, and intensive intensity was constructed based on the peroxidase-like property of 4-mercaptophenylboronic acid (MPBA)-functionalized CuSe nanoprobes (CuSeNPs@MPBA), which improved the detection accuracy and sensitivity. By further integrating a smartphone as an analyzer, quantitative POCT of bacteria was realized with high sensitivity. The limit of detection was as low as 1.25 and 1.01 cfu mL-1 for Staphylococcus aureus and Escherichia coli detection, respectively. Specifically, bacteria can be eliminated with high efficiency due to excellent photothermal property of CuSeNPs@MPBA. The developed multifunctional platform also has advantages of simple operation with low cost, suggesting its high potential for use in food safety, environment monitoring, and clinical applications.

A smartphone integrated ratiometric fluorescent sensor for point-of-care testing of fluoride ions.

In view of the high toxicity and widespread availability, it is of great importance to develop accurate, sensitive, and convenient assays for fluoride ion (F-) detection. Herein, a ratiometric fluorescent system is established for point-of-care testing (POCT) of F- with a smartphone as analyzer. The sensing system of calcein-QDs-Eu3+ contains two fluorescent probes of calcein (green emission) and ZnCdSe/ZnS QDs (red emission). The sensing system only presents red emission in that the calcein emission is quenched due to the combination between calcein and Eu3+. When F- is introduced, the fluorescence of calcein is recovered due to the stronger interaction between F- and Eu3+, which changes the emission from red to green. The ratiometric strategy offers an obvious fluorescence color change of the system, which eliminates interference and improves the detection accuracy. Specifically, the sensing system has excellent selectivity in that Eu3+ is more inclined to bind with F- rather than other anions. The developed assay was further used to prepare a test paper and hydrogel for POCT. To further improve the detection sensitivity and realize quantitative analysis, a smartphone installed with a color scan app is integrated as signal reader and analyzer, which is used for POCT of F- in real samples, showing great application potential in environmental protection and food safety evaluation.

ALKBH3-dependent m1A demethylation of Aurora A mRNA inhibits ciliogenesis.

Primary cilia are antenna-like subcellular structures to act as signaling platforms to regulate many cellular processes and embryonic development. m1A RNA modification plays key roles in RNA metabolism and gene expression; however, the physiological function of m1A modification remains largely unknown. Here we find that the m1A demethylase ALKBH3 significantly inhibits ciliogenesis in mammalian cells by its demethylation activity. Mechanistically, ALKBH3 removes m1A sites on mRNA of Aurora A, a master suppressor of ciliogenesis. Depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation. Moreover, alkbh3 morphants exhibit ciliary defects, including curved body, pericardial edema, abnormal otoliths, and dilation in pronephric ducts in zebrafish embryos, which are significantly rescued by wild-type alkbh3, but not by its catalytically inactive mutant. The ciliary defects caused by ALKBH3 depletion in both vertebrate cells and embryos are also significantly reversed by ectopic expression of Aurora A mRNA. Together, our data indicate that ALKBH3-dependent m1A demethylation has a crucial role in the regulation of Aurora A mRNA, which is essential for ciliogenesis and cilia-associated developmental events in vertebrates.

NudCL2 is an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis.

Primary cilia extending from mother centrioles are essential for vertebrate development and homeostasis maintenance. Centriolar coiled-coil protein 110 (CP110) has been reported to suppress ciliogenesis initiation by capping the distal ends of mother centrioles. However, the mechanism underlying the specific degradation of mother centriole-capping CP110 to promote cilia initiation remains unknown. Here, we find that autophagy is crucial for CP110 degradation at mother centrioles after serum starvation in MEF cells. We further identify NudC-like protein 2 (NudCL2) as a novel selective autophagy receptor at mother centrioles, which contains an LC3-interacting region (LIR) motif mediating the association of CP110 and the autophagosome marker LC3. Knockout of NudCL2 induces defects in the removal of CP110 from mother centrioles and ciliogenesis, which are rescued by wild-type NudCL2 but not its LIR motif mutant. Knockdown of CP110 significantly attenuates ciliogenesis defects in NudCL2-deficient cells. In addition, NudCL2 morphants exhibit ciliation-related phenotypes in zebrafish, which are reversed by wild-type NudCL2, but not its LIR motif mutant. Importantly, CP110 depletion significantly reverses these ciliary phenotypes in NudCL2 morphants. Taken together, our data suggest that NudCL2 functions as an autophagy receptor mediating the selective degradation of mother centriole-capping CP110 to promote ciliogenesis, which is indispensable for embryo development in vertebrates.

Centrosomal protein FOR20 knockout mice display embryonic lethality and left-right patterning defects.

Centrosomal protein FOR20 has been reported to be crucial for essential cellular processes, including ciliogenesis, cell migration, and cell cycle in vertebrates. However, the function of FOR20 during mammalian embryonic development remains unknown. To investigate the in vivo function of the For20 gene in mammals, we generated For20 homozygous knockout mice by gene targeting. Our data reveal that homozygous knockout of For20 results in significant embryonic growth arrest and lethality during gestation, while the heterozygotes show no obvious defects. The absence of For20 leads to impaired left-right patterning of embryos and reduced cilia in the embryonic node. Deletion of For20 also disrupts angiogenesis in yolk sacs and embryos. These results highlight a critical role of For20 in early mammalian embryogenesis.

Inhibition of cell death inducing DNA fragmentation factor-α-like effector c (CIDEC) by tumor necrosis factor-α induces lipolysis and inflammation in calf adipocytes.

Dairy cows with ketosis exhibit signs of pronounced adipose tissue lipolysis and systemic inflammation, both of which exacerbate this metabolic disorder. In nonruminants, CIDEC plays a pivotal role in the formation of large unilocular lipid droplets. The present study aimed to ascertain the role of CIDEC in the lipolytic and inflammatory response of white adipose tissue (WAT) in vivo and in vitro. Subcutaneous adipose tissue and blood samples were collected from 15 healthy cows (blood ß-hydroxybutyrate concentration < 1.2 mM) and 15 cows with clinical ketosis (blood ß-hydroxybutyrate concentration > 3.0 mM) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9). Adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were used for in vitro studies. Isolated adipocytes were treated with 0, 0.1, 1, or 10 ng/mL TNF-α for 3 h, transfected with CIDEC small interfering RNA for 48 h, or transfected with CIDEC overexpression adenovirus for 48 h followed by treatment with TNF-α (0.1 ng/mL) for 3 h. Serum concentrations of fatty acids were greater, and dry matter intake, milk yield, and serum glucose concentrations lower in cows with clinical ketosis. Protein and mRNA abundance of CIDEC were lesser in subcutaneous WAT of clinically ketotic versus healthy cows. Furthermore, the ratio of phosphorylated hormone sensitive lipase (p-LIPE) to LIPE, phosphorylated RELA (p-RELA) to RELA, and protein abundance of PNPLA2 and phosphorylated inhibitor of κBα (p-NFKBIA) were greater in dairy cows with clinical ketosis. The mRNA abundance of proinflammatory cytokines TNFA and IL1B were greater, and the anti-inflammatory cytokine IL10 was lower in WAT of dairy cows with clinical ketosis. In calf adipocytes, exogenous TNF-α (0.1, 1, or 10 ng/mL) decreased protein and mRNA abundance of CIDEC. In addition, exogenous TNF-α or knockdown of CIDEC reduced the secretion of the anti-inflammatory cytokine IL-10, but increased the ratio of p-LIPE to LIPE, p-RELA to RELA, protein abundance of PNPLA2 and p-NFKBIA, glycerol content, and the secretion of IL-1ß in calf adipocytes. Overexpression of CIDEC in TNFα-treated adipocytes attenuated lipolysis and activation of the NF-κB signaling pathway. Overall, these data suggest that inhibition of lipid droplet-associated protein CIDEC by TNF-α contributes to the pronounced lipolysis and inflammation of calf adipocytes, and CIDEC is a relevant target in clinically ketotic cows.

High levels of fatty acids inhibit ß-casein synthesis through suppression of the JAK2/STAT5 and mTOR signaling pathways in mammary epithelial cells of cows with clinical ketosis.

Ketosis is a metabolic disease of dairy cows often characterized by high concentrations of ketone bodies and fatty acids, but low milk protein and milk production. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) and the mechanistic target of rapamycin (mTOR) signaling pathways are central for the regulation of milk protein synthesis. The effect of high levels of fatty acids on these pathways and ß-casein synthesis are unknown in dairy cows with clinical ketosis. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically-ketotic (n = 15) cows. In addition, bovine mammary epithelial cells (BMEC) were treated with fatty acids, methionine (Met) or prolactin (PRL), respectively. In vivo, the serum concentration of fatty acids was greater (P > 0.05) and the percentage of milk protein (P > 0.05) was lower in cows with clinical ketosis. The JAK2-STAT5 and mTOR signaling pathways were inhibited and the abundance of ß-casein was lower in mammary tissue of cows with clinical ketosis (P > 0.05). In vitro, high levels of fatty acids inhibited the JAK2-STAT5 and mTOR signaling pathways (P > 0.05) and further decreased the ß-casein synthesis (P > 0.05) in BMEC. Methionine or PRL treatment, as positive regulators, activated the JAK2-STAT5 and mTOR signaling pathways to increase the ß-casein synthesis. Importantly, the high concentration of fatty acids attenuated the positive effect of Met or PRL on mTOR, JAK2-STAT5 pathways and the abundance of ß-casein (P > 0.05). Overall, these data indicate that the high concentrations of fatty acids that reach the mammary cells during clinical ketosis inhibit mTOR and JAK2-STAT5 signaling pathways, and further suppress ß-casein synthesis.

Mitochondrial membrane protein mitofusin 2 as a potential therapeutic target for treating free fatty acid-induced hepatic inflammation in dairy cows during early lactation.

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and ß-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1ß, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase ß activity, and the abundance of NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.

Low abundance of mitofusin 2 in dairy cows with moderate fatty liver is associated with alterations in hepatic lipid metabolism.

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.

Effects and Mechanism of Blue Light on Monascus in Liquid Fermentation.

The effect of light on Monascus and the underlying mechanism have received a great deal of interest for the industrial application of Monascus pigments. In this study, we have examined the effects of blue light on the culture morphology, mycelium growth, pigments, and citrinin yield of Monascus in liquid-state and oscillation fermentation, and explored the mechanism at a physiological level. It was found that blue light affected the colony morphology, the composition (chitin content), and permeability of the Monascus mycelium cell wall in static liquid culture, which indicates blue light benefits pigments secreting from aerial mycelium to culture medium. In liquid oscillation fermentation, the yields of Monascus pigments in fermentation broth (darkness 1741 U/g, blue light 2206 U/g) and mycelium (darkness 2442 U/g, blue light 1900 U/g) cultured under blue light and darkness are different. The total pigments produced per gram of Monascus mycelium under blue light was also higher (4663 U/g) than that in darkness (4352 U/g). However, the production of citrinin (88 µg/g) under blue light was evidently lower than that in darkness (150 µg/g). According to the degradation of citrinin caused by blue light and hydrogen peroxide, it can be concluded that blue light could degrade citrinin and inhibit the catalase activity of Monascus mycelium, subsequently suppressing the decomposition of hydrogen peroxide, which is the active species that degrades citrinin.

Potential-dependent dynamic fracture of nanoporous gold.

When metallic alloys are exposed to a corrosive environment, porous nanoscale morphologies spontaneously form that can adversely affect the mechanical integrity of engineered structures. This form of stress-corrosion cracking is responsible for the well-known 'season cracking' of brass and stainless steel components in nuclear power generating stations. One explanation for this is that a high-speed crack is nucleated within the porous layer, which subsequently injects into non-porous parent-phase material. We study the static and dynamic fracture properties of free-standing monolithic nanoporous gold as a function electrochemical potential using high-speed photography and digital image correlation. The experiments reveal that at electrochemical potentials typical of porosity formation these structures are capable of supporting dislocation-mediated plastic fracture at crack velocities of 200 m s(-1). Our results identify the important role of high-speed fracture in stress-corrosion cracking and are directly applicable to the behaviour of monolithic dealloyed materials at present being considered for a variety of applications.