Differentiation of Aspartic and Isoaspartic Acid Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y - 1/x ions, while isoAsp-containing peptides yielded more enhanced y - 18/y - 45/y - 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.

Brown tides linked to the unique nutrient profile in coastal waters of Qinhuangdao, China.

Brown tides caused by the pelagophyte Aureococcus anophagefferens have frequently occurred in the Bohai Sea since 2009 and have led to a dramatic collapse of the local scallop culture. To determine why brown tides occurred in the Bohai Sea rather than in other eutrophic coastal waters of China, phytoplankton communities and nutrients were evaluated and nutrient addition experiments were conducted in the Qinhuangdao coastal area. The concentration of dissolved organic nitrogen (DON) was nearly five times higher than that of dissolved inorganic nitrogen (DIN) during brown tides. High levels of phytoplankton biomass and nutrients were observed in the inshore waters, and the patterns of different nutrients were heterogeneous, which could be due to the uneven distribution of pelagophytes and non-brown tide phytoplankton populations (NBTP). The nutrient enrichment results indicated that the growth of the phytoplankton community was nitrogen-limited. Enrichment of DON, especially urea, could promote the growth of pelagophytes during the development stages of the brown tide. In brief, the results of this study imply that the unique nutrient profile (rich in DON but deficient in DIN) could support the outbreak of brown tides in the inshore waters of Qinhuangdao.

Cloning, expression, and bioinformatics analysis of heavy metal resistance-related genes fd-I and fd-II from Acidithiobacillus ferrooxidans.

Five heavy metals were introduced into the bacterial heavy metal resistance tests. The results showed that apparent inhibition effects of Cd2+ and Cu2+ on the growth of Acidithiobacillus ferrooxidans BYSW1 occurred at high concentrations (>0.04 mol l-1). Significant differences (P < 0.001) were both noticed in the expression of two ferredoxin-encoding genes (fd-I and fd-II) related to heavy metal resistance in the presence of Cd2+ and Cu2+ . When exposed to 0.06 mol l-1 Cd2+, the relative expression levels of fd-I and fd-II were about 11 and 13 times as much as those of the control, respectively. Similarly, exposure to 0.04 mol l-1 Cu2+ caused approximate 8 and 4 times higher than those of the control, respectively. These two genes were cloned and expressed in Escherichia coli, and the structures, functions of two corresponding target proteins, i.e. Ferredoxin-I (Fd-I) and Ferredoxin-II (Fd-II), were predicted. The recombinant cells inserted by fd-I or fd-II were more resistant to Cd2+ and Cu2+ compared with wild-type cells. This study was the first investigation regarding the contribution of fd-I and fd-II to enhancing heavy metal resistance of this bioleaching bacterium, and laid a foundation for further elucidation of heavy metal resistance mechanisms caused by Fd.

Food protein glycation: A review focusing on stability and in vitro digestive characteristics of oil/water emulsions.

Recently, increasing studies have shown that the functional properties of proteins, including emulsifying properties, antioxidant properties, solubility, and thermal stability, can be improved through glycation reaction under controlled reaction conditions. The use of glycated proteins to stabilize hydrophobic active substances and to explore the gastrointestinal fate of the stabilized hydrophobic substances has also become the hot spot. Therefore, in this review, the effects of glycation on the structure and function of food proteins and the physical stability and oxidative stability of protein-stabilized oil/water emulsions were comprehensively summarized and discussed. Also, this review sheds lights on the in vitro digestion characteristics and edible safety of emulsion stabilized by glycated protein. It can further serve as a research basis for understanding the role of structural features in the emulsification and stabilization of glycated proteins, as well as their utilization as emulsifiers in the food industry.

Pigment Characterization of the Giant-Colony-Forming Haptophyte Phaeocystis globosa in the Beibu Gulf Reveals Blooms of Different Origins.

The giant-colony-forming haptophyte Phaeocystis globosa has caused several large-scale blooms in the Beibu Gulf since 2011, but the distribution and dynamics of the blooms remained largely unknown. In this study, colonies of P. globosa, as well as membrane-concentrated phytoplankton samples, were collected during eight cruises in the Beibu Gulf from September 2016 to August 2017. Pigments were analyzed by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The pigment 19'-hexanoyloxyfucoxanthin (hex-fuco), generally considered a diagnostic pigment for Phaeocystis, was not detected in P. globosa colonies in the Beibu Gulf, whereas 19'-butanoyloxyfucoxanthin (but-fuco) was found in all colony samples. Moreover, but-fuco in membrane-concentrated phytoplankton samples exhibited a similar distribution pattern to that of P. globosa colonies, suggesting that but-fuco provided the diagnostic pigment for bloom-forming P. globosa in the Beibu Gulf. Based on the distribution of but-fuco in different water masses in the region prior to the formation of intensive blooms, it is suggested that P. globosa blooms in the Beibu Gulf could originate from two different sources. IMPORTANCE Phaeocystis globosa has formed intensive blooms in the South China Sea and even around the world, causing huge social economic losses and environmental damage. However, little is known about the formation mechanism and dynamics of P. globosa blooms. 19'-Hexanoyloxyfucoxanthin (hex-fuco) is often used as the pigment proxy to estimate Phaeocystis biomass, while this is challenged by the giant-colony-forming P. globosa in the Beibu Gulf, which contains only 19'-butanoyloxyfucoxanthin (but-fuco) but not hex-fuco. Using but-fuco as a diagnostic pigment, we traced two different origins of P. globosa blooms in the Beibu Gulf. This study clarifies the development process of P. globosa blooms in the Beibu Gulf, which provides a basis for the early monitoring and prevention of the blooms.

Green Tides in the Yellow Sea Promoted the Proliferation of Pelagophyte Aureococcus anophagefferens.

Harmful algal blooms formed by fast-growing, ephemeral macroalgae have expanded worldwide, yet there is limited knowledge of their potential ecological consequences. Here, we select intense green tides formed by Ulva prolifera in the Yellow Sea, China, to examine the ecological consequences of these blooms. Using 28-isofucosterol in the surface sediment as a biomarker of green algae, we identified the settlement region of massive floating green algae in the area southeast of the Shandong Peninsula in the southern Yellow Sea. The responses of the phytoplankton assemblage from the deep chlorophyll-a maximum layer were then resolved using high-throughput sequencing. We found striking changes in the phytoplankton community in the settlement region after an intensive green tide in 2016, characterized by a remarkable increase in the abundance of the pelagophyte Aureococcus anophagefferens, the causative species of ecosystem disruptive brown tides. Our study strongly suggests that the occurrence of massive macroalgal blooms may promote blooms of specific groups of microalgae through alteration of the marine environment.

Design and synthesis of N-hydroxyalkyl substituted deferiprone: a kind of iron chelating agents for Parkinson's disease chelation therapy strategy.

The blood-brain barrier (BBB) permeability of molecules needs to meet stringent requirements of Lipinski's rule, which pose a difficulty for the rational design of efficient chelating agents for Parkinson's disease chelation therapy. Therefore, the iron chelators employed N-aliphatic alcohols modification of deferiprone were reasonably designed in this work. The chelators not only meet Lipinski's rule for BBB permeability, but also ensure the iron affinity. The results of solution thermodynamics demonstrated that the pFe3+ value of N-hydroxyalkyl substituted deferiprone is between 19.20 and 19.36, which is comparable to that of clinical deferiprone. The results of 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays indicated that the N-hydroxyalkyl substituted deferiprone also possesses similar radical scavenging ability in comparison to deferiprone. Meanwhile, the Cell Counting Kit-8 assays of neuron-like rat pheochromocytoma cell-line demonstrated that the N-hydroxyalkyl substituted deferiprone exhibits extremely low cytotoxicity and excellent H2O2-induced oxidative stress protection effect. These results indicated that N-hydroxyalkyl substituted deferiprone has potential application prospects as chelating agents for Parkinson's disease chelation therapy strategy.

Molecular mechanisms of heavy metals resistance of Stenotrophomonas rhizophila JC1 by whole genome sequencing.

In this study, a higher metal ions-resistant bacterium, Stenotrophomonas rhizophila JC1 was isolated from contaminated soil in Jinchang city, Gansu Province, China. The Pb2+ (120 mg/L) and Cu2+ (80 mg/L) removal rate of the strain reached at 76.9% and 83.4%, respectively. The genome comprises 4268161 bp in a circular chromosome with 67.52% G + C content and encodes 3719 proteins. The genome function analysis showed czc operon, mer operon, cop operon, arsenic detoxification system in strain JC1 were contributed to the removal of heavy metals. Three efflux systems (i.e., RND, CDF, and P-ATPase) on strain JC1 genome could trigger the removal of divalent cations from cells. cAMP pathway and ABC transporter pathway might be involved in the transport and metabolism of heavy metals. The homology analysis exhibited multi-gene families such as ABC transporters, heavy metal-associated domain, copper resistance protein, carbohydrate-binding domain were distributed across 410 orthologous groups. In addition, heavy metal-responsive transcription regulator, thioredoxin, heavy metal transport/detoxification protein, divalent-cation resistance protein CutA, arsenate reductase also played important roles in the heavy metals adsorption and detoxification process. The complete genome data provides insight into the exploration of the interaction mechanism between microorganisms and heavy metals.

High-Quality Carbon Nitride Quantum Dots on Photoluminescence: Effect of Carbon Sources.

Graphitic carbon nitride quantum dots (CNQDs) are a new class of nanomaterial with an extraordinary photoluminescent property. Here, three highly water-soluble and photoluminescent CNQDs are synthesized through a green and facile one-step hydrothermal approach, with urea as the nitrogen source and citric acid and its salts as carbon sources. The photoluminescence (PL) performance demonstrated that the fluorescence emission peak was altered by neither the structures nor the molar ratio of urea to the carbon source. Three highly luminescent CNQDs with a quantum yield of 40% were obtained when the molar ratio of urea to sodium citrate, citric acid, and ammonium citrate was 6:1, 18:1, and 18:1, which have average sizes of 4.1, 4.6, and 6.3 nm, respectively. Moreover, the possibility of using CNQDs as potential probes to determine the concentration of iron is also discussed. The results show that the as-prepared CNQDs has high selectivity for Fe3+ ions. The quenching mechanism of CNQDs by iron is connected with the nitrogen functional groups on the surface of CNQDs. Results showed valuable information about the effects of the carbon source on the PL efficiency, biocompatibility, and metal ion detection properties of CNQDs.

TfOH-Catalyzed [4 + 1] Annulation of p-Quinone Methides with α-Aryl Diazoacetates: Straightforward Access to Highly Functionalized 2,3-Dihydrobenzofurans.

We have developed a methodology for the greatly efficient construction of significant 2,3-dihydrobenzofuran scaffolds bearing a quaternary carbon center at the C2 position by means of [4 + 1] annulation reactions between p-quinone methides and α-aryl diazoacetates as C1 synthons through organocatalysis by readily accessible TfOH catalyst under mild and transition metal-free conditions. This metal-free protocol furnishes an operationally simple and swift process for the free assembly of diverse highly functionalized 2,3-dihydrobenzofurans and also features broad substrate scope, excellent functional group compatibility, and environmental friendliness. Mechanistic investigation suggested that the reaction undergoes a rapid cascade protonation/intermolecular Michael addition/intramolecular substitution process.

Fabrication and photocatalytic activity of graphitic-C3N4 quantum dots-decorated basic zinc carbonate prepared by a co-precipitation method.

Graphitic carbon nitride quantum dots (CNQDs) with a high quantum yield (up to 43%) were synthesized by incorporating the unique organic functional group of barbituric acid into the framework of the carbon nitride structure by supramolecular pre-organization. The CNQDs were introduced onto the surface of basic zinc carbonate (BZC) by co-precipitation. The resulting CNQDs/BZC composite showed that the degradation efficiency of tetracycline was 2.4 times higher than that of ZnO. The Z-scheme mechanism for the as-prepared sample was proposed for the enhanced photocatalytic degradation rate. The ˙O2- and ˙OH radicals played major roles due to the suitable bandgap. Finally, the formation and possible photocatalytic mechanisms of the CNQDs/BZC composite are proposed.

LncRNA NRON promotes M2 macrophage polarization and alleviates atrial fibrosis through suppressing exosomal miR-23a derived from atrial myocytes.

BACKGROUND/PURPOSE: miR-23a is a pro-hypertrophic miRNA that inhibits M2 macrophage polarization. A previous study demonstrated that lncRNA NRON alleviated atrial fibrosis through suppression of M1 macrophages activated by atrial myocytes. This study aimed to determine whether NRON promotes M2 macrophage polarization and alleviates atrial fibrosis through suppressing exosomal miR-23a derived from atrial myocytes. METHODS: Mouse atrial myocytes were transfected with the NRON overexpression vector or empty vector, followed by Ang II treatment. Exosomes were isolated from the treated atrial myocytes and then co-cultured with RAW264.7 macrophages. The culture medium from RAW264.7 macrophages treated as described above was added to mouse atrial fibroblasts for incubation. RESULTS: We found that exosomes derived from Ang II-treated atrial myocytes inhibited M2 macrophage polarization by transferring miR-23a. NFATc3 could directly bind to the miR-23a promoter. Overexpression of NRON inhibited the expression of miR-23a by inhibiting NFATc3 nuclear transport in Ang II-treated atrial myocytes, resulting in a decrease in the level of miR-23a in atrial myocyte-derived exosomes. Meanwhile, exosomes derived from NRON-overexpressing atrial myocytes promoted M2 macrophage polarization and inhibited expression of fibrosis markers in atrial fibroblasts. CONCLUSION: NRON promotes M2 macrophage polarization and alleviates atrial fibrosis through suppressing exosomal miR-23a derived from atrial myocytes.

Pyridine-Diketopyrrolopyrrole-Based Novel Metal-Free Visible-Light Organophotoredox Catalyst for Atom-Transfer Radical Polymerization.

In the field of electronics, organocatalysts are in high demand for use in the synthesis of clean polymers using solar radiation rather than potentially contaminating metals. Combining theoretical design, simulation, and experiments, this work presents a novel, pyridine-diketopyrrolopyrrole (P-DPP)-based metal-free visible-light organophotoredox catalyst (P-DPP). It is effective in the photocontrolled organocatalytic atom-transfer radical polymerization (O-ATRP) of methyl methacrylate (MMA) and styrene. The use of this catalyst and white light-emitting diode (LED) irradiation produces polymers with a cross-linked feature. In O-ATRP, the P-DPP catalyst has an oxidative quenching catalytic mechanism with an excited-state reductive potential of -1.8 V, fluorescence lifetime of 7.5 ns, and radical-cation oxidative potential of 0.45 V. Through molecular simulation, we found that the adjacent pyridine group is key to reducing the alkyl halide initiator and generating radicals, while the diketopyrrolopyrrole core stabilizes the triplet state of the catalyst through intramolecular charge transfer. The findings related to this novel photoredox catalyst will aid in the search for much more effective organophotoredox catalysts for use in controlled radical polymerization. They will also be of value in the fields of polymer chemistry and physics and in various applications.

Hexadentate ß-Dicarbonyl(bis-catecholamine) Ligands for Efficient Uranyl Cation Decorporation: Thermodynamic and Antioxidant Activity Studies.

The special linear dioxo cation structure of the uranyl cation, which relegates ligand coordination to an equatorial plane perpendicular to the O═U═O vector, poses an unusual challenge for the rational design of efficient chelating agents. Therefore, the planar hexadentate ligand rational design employed in this work incorporates two bidentate catecholamine (CAM) chelating moieties and a flexible linker with a ß-dicarbonyl chelating moiety (ß-dicarbonyl(CAM)2 ligands). The solution thermodynamics of ß-dicarbonyl(CAM)2 with a uranyl cation was investigated by potentiometric and spectrophotometric titrations. The results demonstrated that the pUO22+ values are significantly higher than for the previously reported TMA(2Li-1,2-HOPO)2, and efficient chelation of the uranyl cation was realized by the planar hexadentate ß-dicarbonyl(CAM)2. The efficient chelating ability of ß-dicarbonyl(CAM)2 was attributed to the presence of the more flexible ß-dicarbonyl chelating linker and planar hexadentate structure, which favors the geometric arrangement between ligand and uranyl coordinative preference. Meanwhile, ß-dicarbonyl(CAM)2 also exhibits higher antiradical efficiency in comparison to butylated hydroxyanisole. These results indicated that ß-dicarbonyl(CAM)2 has potential application prospects as a chelating agent for efficient chelation of a uranyl cation.

Large-area snow-like MoSe2 monolayers: synthesis, growth mechanism, and efficient electrocatalyst application.

This study explores the large-area synthesis of controllable morphology, uniform, and high-quality monolayer. MoSe2 is essential for its potential application in optoelectronics, photocatalysis, and renewable energy sources. In this study, we successfully synthesized snow-like MoSe2 monolayers using a simple chemical vapor deposition method. Results reveal that snow-like MoSe2 is a single crystal with a hexagonal structure, a thickness of ∼0.9 nm, and a lateral dimension of up to 20 µm. The peak position of the photoluminescence spectra is ∼1.52 eV corresponding to MoSe2 monolayer. The growth mechanism of the snow-like MoSe2 monolayer was investigated and comprised a four-step process during growth. Finally, we demonstrate that the snow-like MoSe2 monolayers are ideal electrocatalysts for hydrogen evolution reactions (HERs), reflected by a low Tafel slope of ∼68 mV/decade. Compared with the triangular-shaped MoSe2 monolayer, the hexangular snow-like shape with plentiful edges is superior for perfect electrocatalysts for HERs or transmission devices of optoelectronic signals.

High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms.

Cysteine racemization on IgG heavy and light chains.

Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of D-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible ß-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide.

IgG1 thioether bond formation in vivo.

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.

Fabrication of a highly b-oriented MFI-type zeolite film by the Langmuir-Blodgett method.

sec-Butanol-modified rounded-coffin-shaped silicalite-1 (SL) microcrystals were assembled into a compact and highly b-oriented monolayer extending over the centimeter scale via the Langmuir-Blodgett (LB) technique. For comparison, methanol- or ethanol-modified SL microcrystals could not float and were compressed into a dense film in an LB trough. Subsequently, highly b-oriented MFI films with a thickness of ∼1.5 µm were successfully obtained on the solid substrates by secondary growth of the LB monolayer using tetrapropylammonium hydroxide (TPAOH) as the structure-directing agent. The electrochemical experiments confirmed that the prepared films were defect-free. In general, the LB method is a highly controllable and reproducible method of organizing anisotropic zeolite crystals with a preferred orientation over a relatively large surface area. The LB technique could be further applied as an effective platform for the oriented assembly of different types of zeolite particles and the growth of variously oriented zeolite films.

In situ controlled release of rhBMP-2 in gelatin-coated 3D porous poly(ε-caprolactone) scaffolds for homogeneous bone tissue formation.

In tissue engineering, incorporation of bone morphogenetic protein-2 (BMP-2) into biomaterial scaffolds is an attractive strategy to stimulate bone repair. However, suboptimal release of BMP-2 remains a great concern, which may cause unfavorable bone formation as well as severe inflammation. In this study, genipin-cross-linked gelatin entrapped with recombinant human BMP-2 (rhBMP-2) was exploited to decorate the interior surface of three-dimensional porous poly(ε-caprolactone) (PCL) scaffolds. With gelatin-coating, PCL scaffolds demonstrated enhanced water uptake and improved compressive moduli. Intriguingly, a unique release profile of rhBMP-2 composed of a transient burst release followed by a sustained release was achieved in coated scaffolds. These coated scaffolds well supported growth and osteogenesis of human mesenchymal stem cells (hMSCs) in vitro, indicating the retaining of rhBMP-2 bioactivity. When hMSCs-seeded scaffolds were implanted subcutaneously in nude mice for 4 weeks, better bone formation was observed in gelatin/rhBMP-2-coated scaffolds. Specifically, the spatial distribution of newly formed bone was more uniform in gelatin-coated scaffolds than in uncoated scaffolds, which displayed preferential bone formation at the periphery. These results collectively demonstrated that gelatin-coating of porous PCL scaffolds is a promising approach for delivering rhBMP-2 to stimulate improved bone regeneration.