Near-infrared fluorescent probe based on the regulatory dye pKa for imaging of H2S in rice roots and living cells.

Hydrogen sulfide (H2S) is a key factor in various biological processes such as plant grow and its response to environmental stress. Here, we develop a novel near-infrared (NIR) fluorescent probe for detecting hydrogen sulfide based on the regulatory NIR dye pKa values. After triggering the H2S substitution response, probe A with introducing the cyano moiety not only exhibits a significant near-infrared emission (Emax: 724 nm) response in physiological environments, but also shows a fast response, high selectivity, and sensitivity (LOD as 0.52 µM). In addition, probe A with low biological cytotoxicity is successfully used for imaging detection of cellular exogenous and endogenous hydrogen sulfide. More importantly, in situ imaging of probe A tracks the H2S fluctuations in the rice root system and its response to environmental stress. Hence, this work offers a new NIR fluorescence imaging monitoring tool for hydrogen sulfide in biological systems.

Implementation and assessment of an end-to-end Open Science & Data Collaborations program.

As research becomes more interdisciplinary, fast-paced, data-intensive, and collaborative, there is an increasing need to share data and other research products in accordance with Open Science principles. In response to this need, we created an Open Science & Data Collaborations (OSDC) program at the Carnegie Mellon University Libraries that provides Open Science tools, training, collaboration opportunities, and community-building events to support Open Research and Open Science adoption. This program presents a unique end-to-end model for Open Science programs because it extends open science support beyond open repositories and open access publishing to the entire research lifecycle. We developed a logic model and a preliminary assessment metrics framework to evaluate the impact of the program activities based on existing data collected through event and workshop registrations and platform usage. The combination of these evaluation instruments has provided initial insight into our service productivity and impact. It will further help to answer more in-depth questions regarding the program impact, launch targeted surveys, and identify priority service areas and interesting Open Science projects.

Hardening Mechanism of Coral Aggregates in Cement-Based Systems.

In terms of the infrastructure construction near coral reefs, native coral aggregates have been widely implemented as alternative efficient building materials to prepare the "coral concrete". This study focused on the mechanical properties and hardening mechanism of coral particles under cement-based systems. Firstly, coral particles were divided into two categories: coral biological debris particles (calcium sand) and coral parent rock particles (limestone). Subsequently, several elementary laboratory tests were conducted to compare the physical and chemical properties of the samples. The results demonstrate that the specific surface area and open pores of coral particles are bigger than those of quartz sand. Moreover, the water absorption rate of debris and parent rock particles reach 9.9% and 22%, respectively. To further examine the hardening mechanism of coral particles, we carried out particle crushing strength tests, compressive strength tests and nanoindentation tests. Regardless of the tested groups and particle categories, the results show that the wrapped cement slurry universally demonstrated the successful enhancement of crushing strength σ0,d. Particularly, in the size range from 1.18-2.36 mm, wrapped particles of debris and parent rock both reached unusually high σ0,d values: 10.14 MPa and 8.74 MPa, respectively. However, in the size range from 9.5 mm to 16 mm, the σ0,d values of wrapped debris and parent rock reached 4.75 MPa and 3.18 MPa, respectively. According to the nanoindentation tests, the sub-microscopic strength of ITZs was enhanced to more than 1 GPa, which is higher than that of conventional concrete, in terms of the sample with 28-day maintenance. In conclusion, this study has provided a further basis for studying coral concrete material and its hardening mechanism.

Synchronous Construction of the Hierarchical Pores and High Hydrophobicity of Stable Metal-Organic Frameworks through a Dual Coordination-Competitive Strategy.

Hierarchical-pore construction and functionalities are critical to further extend the applications of some stable MOFs, such as water remediation, fuel purification, oil/water separation, and self-cleaning, which are rarely achieved simultaneously. Herein, we demonstrate a method of synchronously constructing high-hydrophobicity Zr-based metal-organic frameworks with hierarchical pores (HP-UiO-66) through a dual coordination-competitive strategy. The addition of alkanoic acids and Zn2+ ions as coordination-competitors could reduce the coordinative degree between the ligand and Zr4+ ions to effectively induce defect formation. The resulting unsaturated Zr4+ ions could fully combine with the existing alkanoic acid with a long chain to afford HP-UiO-66 with high-hydrophobicity characteristics. In addition, the particle size of pristine UiO-66 could be adjusted effectively from around 280 to 120 nm using different alkanoic acids when Zn2+ ions are not added. This study provided a simple way for effectively controlling the morphology and structure of UiO-66 at the same time. Moreover, this kind of high-hydrophobicity HP-UiO-66 showed potential applications in oil/water separation and selective adsorption of organic mixtures.

Partitioning of MLX-Family Transcription Factors to Lipid Droplets Regulates Metabolic Gene Expression.

Lipid droplets (LDs) store lipids for energy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in macrophages, a cell type involved in metabolic diseases. Among ∼550 identified screen hits is MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates metabolic processes. We show that MLX and glucose-sensing family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs accumulate in cells, these transcription factors bind to LDs, reducing their availability for transcriptional activity and attenuating the response to glucose. Conversely, the absence of LDs results in hyperactivation of MLX target genes. Our findings uncover a paradigm for a lipid storage response in which binding of MLX transcription factors to LD surfaces adjusts the expression of metabolic genes to lipid storage levels.

Substituent Effects of Phenyl Group on Silylene Bridge in Stereospecific Polymerization of Propylene with C1-Symmetric Ansa-Silylene(fluorenyl)(amido) Dimethyl Titanium Complexes.

A C1-symmetric (methylphenyl)silylene-bridged (fluorenyl)(naphthylamido) titanium complex (1) and (diphenyl)silylene-bridged (fluorenyl)(naphthylamido) titanium complex (2) were synthesized and characterized by ¹H NMR, element analysis, and X-ray crystal analysis. The coordination mode of the fluorenyl ligand to the titanium metal is an η³ manner in each complex. These complexes were applied for propylene polymerization using dried modified methyaluminoxane (dMMAO) as a cocatalyst under different propylene pressures in a semi batch-type method. The catalytic activity was strongly dependent on the structure of the complex and the propylene pressure, where complex 1 exhibited the highest activity (600 kg mol-1·h-1) under a propylene pressure of 8.0 atm to produce high molecular weight polypropylene. The polypropylenes obtained were syndiotactic-rich with an rr value of 0.50, indicating that the silylene bridge was not efficient for the isospecificity of a constrained geometry catalyst (CGC). The mechanical properties of the resulting polymers depended on their microstructure.

Functional Contribution of the Spastic Paraplegia-Related Triglyceride Hydrolase DDHD2 to the Formation and Content of Lipid Droplets.

Deleterious mutations in the serine lipase DDHD2 are a causative basis of complex hereditary spastic paraplegia (HSP, subtype SPG54) in humans. We recently found that DDHD2 is a principal triglyceride hydrolase in the central nervous system (CNS) and that genetic deletion of this enzyme in mice leads to ectopic lipid droplet (LD) accumulation in neurons throughout the brain. Nonetheless, how HSP-related mutations in DDHD2 relate to triglyceride metabolism and LD formation remains poorly understood. Here, we have characterized a set of HSP-related mutations in DDHD2 and found that they disrupt triglyceride hydrolase activity in vitro and impair the capacity of DDHD2 to protect cells from LD accumulation following exposure to free fatty acid, an outcome that was also observed with a DDHD2-selective inhibitor. We furthermore isolated and characterized LDs from brain tissue of DDHD2-/- mice, revealing that they contain both established LD-associated proteins identified previously in other organs and CNS-enriched proteins, including several proteins with genetic links to human neurological disease. These data, taken together, indicate that the genetic inactivation of DDHD2, as caused by HSP-associated mutations, substantially perturbs lipid homeostasis and the formation and content of LDs, underscoring the importance of triglyceride metabolism for normal CNS function and the key role that DDHD2 plays in this process.

Protective Effects of an Ancient Chinese Kidney-Tonifying Formula against H2O2-Induced Oxidative Damage to MES23.5 Cells.

Oxidative damage plays a critical role in the etiology of neurodegenerative disorders including Parkinson's disease (PD). In our study, an ancient Chinese kidney-tonifying formula, which consists of Cistanche, Epimedii, and Polygonatum cirrhifolium, was investigated to protect MES23.5 dopaminergic neurons against hydrogen peroxide- (H2O2-) induced oxidative damage. The damage effects of H2O2 on MES23.5 cells and the protective effects of KTF against oxidative stress were evaluated using MTT assay, transmission electron microscopy (TEM), immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The results showed that cell viability was dramatically decreased after a 12 h exposure to 150 µM H2O2. TEM observation found that the H2O2-treated MES23.5 cells presented cellular organelle damage. However, when cells were incubated with KTF (3.125, 6.25, and 12.5 µg/ml) for 24 h after H2O2 exposure, a significant protective effect against H2O2-induced damage was observed in MES23.5 cells. Using ICC, we found that KTF inhibited the reduction of the tyrosine hydroxylase (TH) induced by H2O2, upregulated the mRNA and protein expression of HO-1, CAT, and GPx-1, and downregulated the expression of caspase 3. These results indicated that KTF may provide neuron protection against H2O2-induced cell damage through ameliorating oxidative stress, and our findings provide a new potential strategy for the prevention and treatment of Parkinson's disease.

Living Polymerization of Propylene with ansa-Dimethylsilylene(fluorenyl)(cumylamido) Titanium Complexes.

A series of ansa-silylene(fluorenyl)(amido) titanium complexes (1a⁻1c, 2a, and 2b) bearing various substituents on the amido and fluorenyl ligands are synthesized and characterized by elemental analysis, ¹H NMR, and single crystal X-ray analysis. The coordination mode of the fluorenyl ligand to the titanium metal is η³ manner in each complex. The propylene polymerization is conducted with these complexes at 0 and 25 °C in a semi batch-type method, respectively. The catalytic activity of 1a⁻1c bearing cumyl-amido ligand is much higher than that of 2a and 2b bearing naphthyl group in amido ligand. High molecular weight polypropylenes are obtained with narrow molecular weight distribution, suggesting a living nature of these catalytic systems at 0 °C. The polymers produced are statistically atactic, regardless of the structure of the complex and the polymerization temperature.

Seipin is required for converting nascent to mature lipid droplets.

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

Ecotoxicity monitoring and bioindicator screening of oil-contaminated soil during bioremediation.

A series of toxicity bioassays was conducted to monitor the ecotoxicity of soils in the different phases of bioremediation. Artificially oil-contaminated soil was inoculated with a petroleum hydrocarbon-degrading bacterial consortium containing Burkholderia cepacia GS3C, Sphingomonas GY2B and Pandoraea pnomenusa GP3B strains adapted to crude oil. Soil ecotoxicity in different phases of bioremediation was examined by monitoring total petroleum hydrocarbons, soil enzyme activities, phytotoxicity (inhibition of seed germination and plant growth), malonaldehyde content, superoxide dismutase activity and bacterial luminescence. Although the total petroleum hydrocarbon (TPH) concentration in soil was reduced by 64.4%, forty days after bioremediation, the phytotoxicity and Photobacterium phosphoreum ecotoxicity test results indicated an initial increase in ecotoxicity, suggesting the formation of intermediate metabolites characterized by high toxicity and low bioavailability during bioremediation. The ecotoxicity values are a more valid indicator for evaluating the effectiveness of bioremediation techniques compared with only using the total petroleum hydrocarbon concentrations. Among all of the potential indicators that could be used to evaluate the effectiveness of bioremediation techniques, soil enzyme activities, phytotoxicity (inhibition of plant height, shoot weight and root fresh weight), malonaldehyde content, superoxide dismutase activity and luminescence of P. phosphoreum were the most sensitive.

Surface-enhanced fluorescent immunoassay on 2D silver nanotriangles array.

Long range ordered silver nanotriangles array was fabricated for surface-enhanced fluorescent immunoassay in this paper. By polystyrene (PS) microspheres based LB template method, the silver nanotriangle array with about 100 nm in height was constructed on the surface of glass slide. On the surface of Ag nanotriangles array, the immune reaction of antigens and labeled antibodies was carried out. Based on the interaction of fluorophores from antibodies with the plasmon resonance from Ag nanotriangles and the enrichment effect of this patterned array, 3.11 times enhancement of the fluorescent intensity of the target antibodies was obtained. According to the fitting curve of fluorescent intensities and logarithmic concentrations of labeled antibodies from 100 pg/mL to 10 µg/mL, it concludes that the limit of detection by this Ag nanotriangles array for immune complex is 100 pg/mL. Due to the advantages of high sensitivity, good reproducibility, and convenient fabrication, the 2D silver nanotriangles array could be an exciting platform for bioassays in proteomics, drug discovery and diagnostics.

Analysis of lipid droplets in hepatocytes.

The liver plays an important role in triacylglycerol (TG) metabolism. It can store large amounts of TG in cytosolic lipid droplets (CLDs), or it can package TG into very-low density lipoproteins (VLDL) that are secreted from the cell. TG packaged into VLDL is derived from TG stored within the endoplasmic reticulum in lumenal lipid droplets (LLDs). Therefore, the liver contains at least three kinds of LDs that differ in their protein composition, subcellular localization, and function. Hepatic LDs undergo tremendous changes in their size and protein composition depending on the energetic (fasting/feeding) and pathological (viral infection, nonalcoholic fatty liver disease, etc.) states. It is crucial to develop methodologies that allow the isolation and analyses of the various hepatic LDs in order to gain insight into the differential metabolism of these important lipid storage/transport particles in health and disease. Here, we present detailed protocols for the isolation and analysis of CLDs and LLDs and for monitoring CLD dynamics.

Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Lipin-1γ isoform is a novel lipid droplet-associated protein highly expressed in the brain.

Lipin-1 proteins are phosphatidic acid phosphatases (PAPs) catalyzing the conversion from phosphatidic acid (PA) to diacylglycerol (DG). Two alternative splicing isoforms, lipin-1α and -1ß, are localized at different subcellular compartments. A third splicing isoform, lipin-1γ was recently cloned and its subcellular localization is unknown. Here, we demonstrate that lipin-1γ is localized to lipid droplets (LDs), an association mediated by a hydrophobic, lipin-1γ-specific domain. Additional expression of lipin-1γ altered LD morphology without affecting the triacylglycerol (TG) level. In human tissues, lipin-1γ is the main lipin-1 isoform expressed in normal human brain, suggesting a specialized role in regulating brain lipid metabolism.

Hepatic autophagy mediates endoplasmic reticulum stress-induced degradation of misfolded apolipoprotein B.

UNLABELLED: Induction of endoplasmic reticulum (ER) stress was previously shown to impair hepatic apolipoprotein B100 (apoB) production by enhancing cotranslational and posttranslational degradation of newly synthesized apoB. Here, we report the involvement of autophagy in ER stress-induced degradation of apoB and provide evidence for a significant role of autophagy in regulating apoB biogenesis in primary hepatocyte systems. Induction of ER stress following short-term glucosamine treatment of McA-RH7777 cells resulted in significantly increased colocalization of apoB with green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3), referred to as apoB-GFP-LC3 puncta, in a dose-dependent manner. Colocalization with this autophagic marker correlated positively with the reduction in newly synthesized apoB100. Treatment of McA-RH7777 cells with 4-phenyl butyric acid, a chemical ER stress inhibitor, prevented glucosamine- and tunicamycin-induced increases in GRP78 and phosphorylated eIF2α, rescued newly synthesized [(35) S]-labeled apoB100, and substantially blocked the colocalization of apoB with GFP-LC3. Autophagic apoB degradation was also observed in primary rat and hamster hepatocytes at basal conditions as well as upon the induction of ER stress. In contrast, this pathway was inactive in HepG2 cells under ER stress conditions, unless proteasomal degradation was blocked with N-acetyl-leucinyl-leucinyl-norleucinal and the medium was supplemented with oleate. Transient transfection of McA-RH7777 cells with a wild-type protein kinase R-like ER kinase (PERK) complementary DNA resulted in dramatic induction of apoB autophagy. In contrast, transfection with a kinase inactive mutant PERK gave rise to reduced apoB autophagy, suggesting that apoB autophagy may occur via a PERK signaling-dependent mechanism. CONCLUSION: Taken together, these data suggest that induction of ER stress leads to markedly enhanced apoB autophagy in a PERK-dependent pathway, which can be blocked with the chemical chaperone 4-phenyl butyric acid. ApoB autophagy rather than proteasomal degradation may be a more pertinent physiological mechanism regulating hepatic lipoprotein production in primary hepatocytes.

Altered lipid droplet dynamics in hepatocytes lacking triacylglycerol hydrolase expression.

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

Loss of TGH/Ces3 in mice decreases blood lipids, improves glucose tolerance, and increases energy expenditure.

Excessive accumulation of triacylglycerol in peripheral tissues is tightly associated with obesity and has been identified as an independent risk factor for insulin resistance, type 2 diabetes, and cardiovascular complications. Here we show that ablation of carboxylesterase 3 (Ces3)/triacylglycerol hydrolase (TGH) expression in mice (Tgh(-/-)) results in decreased plasma triacylglycerol, apolipoprotein B, and fatty acid levels in both fasted and fed states. Despite the attenuation of very low-density lipoprotein secretion, TGH deficiency does not increase hepatic triacylglycerol levels. Tgh(-/-) mice exhibit increased food intake, respiratory quotient, and energy expenditure without change in body weight. These metabolic changes are accompanied by improved insulin sensitivity and glucose tolerance. Tgh(-/-) mice have smaller sized pancreatic islets but maintain normal glucose-stimulated insulin secretion. These studies demonstrate the potential of TGH as a therapeutic target for lowering blood lipid levels.

Proteomic and lipid characterization of apolipoprotein B-free luminal lipid droplets from mouse liver microsomes: implications for very low density lipoprotein assembly.

The assembly of very low density lipoproteins involves the formation of a primordial, poorly lipidated apoB-containing particle in the endoplasmic reticulum, followed by the addition of neutral lipid from luminal lipid droplets (LLD). However, the lipid and protein compositions of LLD have not been determined. We have isolated LLD from mouse liver microsomes and analyzed their lipid and protein compositions. LLD are variably sized particles relatively poor in triacylglycerol (TG) content when compared with the lipid composition of cytosolic lipid droplets (CLD). They are devoid of apoB, adipophilin, and albumin but contain numerous proteins different from those found on CLD, including TG hydrolase (TGH), carboxylesterase 1 (Ces1), microsomal triglyceride transfer protein (MTP), and apoE. Ectopic expression of TGH in McArdle RH7777 hepatoma cells resulted in decreased cellular TG levels, demonstrating a role for TGH in the mobilization of hepatic neutral lipid stores. The isolation and characterization of LLD provide new supporting evidence for the two-step assembly of very low density lipoproteins.