Lijuan Zhang

Excessive N fertilization in intensive agricultural areas of China has resulted in serious environmental problems because of atmospheric, soil, and water enrichment with reactive N of agricultural origin. This study examines grain yields and N loss pathways using a synthetic approach in 2 of the most intensive double-cropping systems in China: waterlogged rice/upland wheat in the Taihu region of east China versus irrigated wheat/rainfed maize on the North China Plain. When compared with knowledge-based optimum N fertilization with 30-60% N savings, we found that current agricultural N practices with 550-600 kg of N per hectare fertilizer annually do not significantly increase crop yields but do lead to about 2 times larger N losses to the environment. The higher N loss rates and lower N retention rates indicate little utilization of residual N by the succeeding crop in rice/wheat systems in comparison with wheat/maize systems. Periodic waterlogging of upland systems caused large N losses by denitrification in the Taihu region. Calcareous soils and concentrated summer rainfall resulted in ammonia volatilization (19% for wheat and 24% for maize) and nitrate leaching being the main N loss pathways in wheat/maize systems. More than 2-fold increases in atmospheric deposition and irrigation water N reflect heavy air and water pollution and these have become important N sources to agricultural ecosystems. A better N balance can be achieved without sacrificing crop yields but significantly reducing environmental risk by adopting optimum N fertilization techniques, controlling the primary N loss pathways, and improving the performance of the agricultural Extension Service.

Sequencing and de novo assembly of seven wild relatives of soybean yields insights relevant to crop domestication and improvement. Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

Electrocatalytic CO2 reduction (ECR) with rationally designed electrocatalysts is a promising strategy to reduce CO2 emission and produce value-added products. Reactive sites of heterogeneous catalysts usually lie on the surface and subsurface, which allow improvement of the catalytic property by engineering the surface atoms. Defects of an electrocatalyst, such as dopants, atom vacancies, and grain boundaries, have potential to enable unconventional adsorption behaviors and chemical activities of reactants on the catalyst surface, and selectively enhance the stability of specific intermediates and corresponding ECR pathways. Moreover, the interface between two different electrocatalyst components can also stabilize active surface catalytic sites and enable their synergetic effects. In this review, we summarize how surface defects and interface can be rationally designed and functioned in ECR catalysts, and how these atomic-level controlling approaches help to promote efficiency and selectivity. The challenges and prospects are also discussed to suggest the future designs of ECR catalysts.

Highly ordered mesoporous carbons and silicas with ultralarge accessible pores have been successfully synthesized by using laboratory-made poly(ethylene oxide)-b-polystyrene (PEO-b-PS) diblock copolymers as templates via the evaporation-induced self-assembly (EISA) approach. Resols and tetraethyl orthosilicate (TEOS) serve as carbon and silica precursors, respectively. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) measurements show that the mesoporous carbons (denoted as C-FDU-18) possess face centered cubic closed-packing (fcc) mesostructure (Fm3̄m) with large-domain ordering. N2 sorption isotherms reveal a large mesopore at the mean value of 22.6 nm with a narrow pore-size distribution. Mesoporous silicas (Si-FDU-18) also display a highly ordered fcc closed-packing mesostructure with an ultralarge unit cell (a = 54.6 nm). A hydrothermal recrystallization was introduced for the first time to produce micropores in thick silica walls (∼7.7 nm) and thus to generate ultralarge accessible mesopores (30.8 nm). Notably, the amphiphilic diblock copolymer with high molecular weight (PEO125-PS230, 29700 g mol-1) in this report was prepared via a simple method of atom transfer radical polymerization (ATRP). It can be easily available for chemists even without any experience in polymer synthesis.

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including α-helical, β-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two α-helical peptides V681n and V25p, the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the β-sheet peptide polyphemusin, intermediate for α-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes. A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including α-helical, β-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two α-helical peptides V681n and V25p, the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the β-sheet peptide polyphemusin, intermediate for α-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes. minimal inhibitory concentration 1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine phosphatidylglycerol from egg yolk l-α-phosphatidy-dl-choline from egg yolk cardiolipin 1-palmitoyl-2-[6-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-caproyl]-l-α-phosphatidylcholine dansyl phosphatidylethanolamine Cationic antimicrobial peptides are important components of innate immunity, and their distribution throughout the animal kingdom is widespread, including bacteria, fungi, plants, insects, birds, crustaceans, amphibians, and mammals (1Boman H.G. Scand. J. Immunol. 1996; 43: 475-482Crossref PubMed Scopus (59) Google Scholar, 2Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1998; 10: 41-44Crossref PubMed Scopus (339) Google Scholar). The naturally occurring peptides are generally 12- to 50-amino acids-long and folded into a variety of different structures, including α-helices, β-sheets, extended helices, and loops (3Hancock R.E.W. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). Despite this structural variation and extensive sequence variability, most antimicrobial cationic peptides share two unique features, in that they are polycationic, with a net positive charge of more than +2, and fold into amphipathic structures, with both a hydrophobic and a hydrophilic domain (3Hancock R.E.W. Lancet. 1997; 349: 418-422Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar). These characteristics allow them to interact with the negatively charged surface molecule lipopolysaccharide of Gram-negative bacteria and to interact with and insert into the negatively charged cytoplasmic membranes of most bacteria. Antimicrobial peptides exhibit rapid killing, often within minutes in vitro, and a broad spectrum of killing activity against various targets, including Gram-negative and Gram-positive bacteria, fungi, enveloped viruses, parasites, and even tumor cells (4Maloy W.L. Kari U.P. Biopolymers. 1995; 37: 105-122Crossref PubMed Scopus (463) Google Scholar, 5Hancock R.E.W. Lehrer R. Trends Biotechnol. 1998; 16: 82-88Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar). Therefore, improved variants of naturally occurring antimicrobial peptides may provide a feasible alternative to conventional antibiotics, especially because of the emergence of resistant bacterial strains worldwide (6Hancock R.E.W. Expert Opin. Investig. Drugs. 2000; 9: 1723-1729Crossref PubMed Scopus (140) Google Scholar, 7Hancock R.E.W. Drugs. 1999; 57: 469-473Crossref PubMed Scopus (120) Google Scholar). Although there are many studies addressing the issue, the molecular mechanism underlying antimicrobial peptide-mediated cell death is still a matter of debate. A considerable body of data indicates that most antimicrobial peptides interact with the cytoplasmic membrane rather than by interacting with a specific protein receptor (8Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1155) Google Scholar). For example, certain membrane-active peptides, such as cecropins, magainins, and melittins, etc., have been demonstrated to permeabilize model membrane systems, cause leakage of fluorescent dyes from unilamellar liposomes, or induce ion transport across lipid bilayers (9Dathe M. Wieprecht T. Biochim. Biophys. Acta. 1999; 1462: 71-87Crossref PubMed Scopus (636) Google Scholar, 10Cafiso D.S. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 141-165Crossref PubMed Scopus (250) Google Scholar). This has lead to the general conclusion that lysis or leakage of essential molecules due to formation of channels in the cytoplasmic membrane is the mechanism of killing. However, such model membrane experiments can sometimes be criticized, because they use very high peptide/lipid ratios. Consistent with this, intact cell experiments are generally inconsistent with the concept of peptide lysis of bacteria (11Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (411) Google Scholar), and even the breakdown of the cytoplasmic membrane permeability barrier at the minimal inhibitory concentration (MIC)1 has been questioned (11Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (411) Google Scholar, 12Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (634) Google Scholar). It is noteworthy that if a high enough concentration of virtually any cationic peptide is used, the cytoplasmic membrane does become leaky, but no formal relationship between killing and permeabilization has been established for many peptides. Although some reports have shown that peptides can become oriented perpendicular to the bilayer at very high peptide/lipid ratios, most studies from NMR, Raman, and fluorescence measurements indicated that cationic peptides initially bind parallel to the lipid bilayer, probably around the interface of head groups and fatty acyl chains (13Bechinger B. Zasloff M. Opella S.J. Protein Sci. 1993; 2: 2077-2084Crossref PubMed Scopus (344) Google Scholar, 14Bechinger B. Zasloff M. Opella S.J. Biophys. J. 1998; 74: 981-987Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Matsuzaki K. Nakayama M. Fukui M. Otaka A. Funakoshi S. Fujii N. Bessho K. Miyajima K. Biochemistry. 1993; 32: 11704-11710Crossref PubMed Scopus (70) Google Scholar, 16Matsuzaki K. Murase O. Tokuda H. Funakoshi S. Fujii N. Miyajima K. Biochemistry. 1994; 33: 3342-3349Crossref PubMed Scopus (291) Google Scholar). Thus it has been proposed that such interfacial peptides might enhance membrane permeabilization by disrupting lipid organization and packing instead of forming a formal pore (17Shai Y. Biochim. Biophys. Acta. 1999; 1462: 55-70Crossref PubMed Scopus (1591) Google Scholar). Conversely it has been suggested that peptides form informal “aggregate channels” containing lipid and peptide and that using this as an intermediate, some peptides can translocate across the cytoplasmic membrane (16Matsuzaki K. Murase O. Tokuda H. Funakoshi S. Fujii N. Miyajima K. Biochemistry. 1994; 33: 3342-3349Crossref PubMed Scopus (291) Google Scholar, 18Hancock R.E.W. Chapple D.S. Antimicrob. Agents Chemother. 1999; 43: 1317-1323Crossref PubMed Google Scholar). Therefore, inhibition of DNA, RNA, and/or protein biosynthesis has also been proposed as an alternative mechanism resulting in cell death (11Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (411) Google Scholar, 19Lehrer R.I. Barton A. Daher K.A. Harwig S.S. Ganz T. Selsted M.E. J. Clin. Invest. 1989; 84: 553-561Crossref PubMed Scopus (590) Google Scholar, 20Park C.B. Kim H.S. Kim S.C. Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (698) Google Scholar). Regardless of this debate, it is agreed by all researchers that the interaction of most peptides with the membrane, involving charge/charge and hydrophobic interactions, is a necessary precursor to cell death (21Sitaram N. Nagaraj R. Biochim. Biophys. Acta. 1999; 1462: 29-54Crossref PubMed Scopus (301) Google Scholar). The interactions of antimicrobial cationic peptides with model phospholipid membranes have been extensively studied for more than two decades. However, the molecular mechanism of peptide-membrane interaction is still controversial. Studies on the frog skin peptide magainin 2 indicate that this peptide causes lipid flip-flop, coupled with pore formation and peptide translocation across the bilayer (22Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1996; 35: 11361-11368Crossref PubMed Scopus (634) Google Scholar). This finding is interesting and has shed some light on the molecular mechanism of peptide-membrane interaction. Unfortunately, such studies have been largely limited to magainin and structurally related peptides, and most experiments were carried out using high peptide/lipids ratios of 1:10 to 1:50 (22Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1996; 35: 11361-11368Crossref PubMed Scopus (634) Google Scholar, 23Kobayashi S. Takeshima K. Park C.B. Kim S.C. Matsuzaki K. Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (185) Google Scholar). In the present study we have tried to work at lower peptide/lipid ratios and asked whether peptides with different structures and activities have the same mechanism of action. To provide structural diversity we have selected peptides from two previous structure-activity relationship studies. One group included α-helical peptides derived from a cecropin-melittin hybrid without proline or with one or two prolines, represented here by V681n, V25p, and V8pp, respectively (24Zhang L. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 8102-8111Crossref PubMed Scopus (135) Google Scholar). The other group included the extended, boat-shaped, bovine neutrophil peptide indolicidin and its two structural variants CP11CN and CP10A, with improved Gram-negative and broad spectrum activity, respectively (11Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (411) Google Scholar,25Rozek A. Friedrich C.L. Hancock R.E.W. Biochemistry. 2000; 39: 15765-15774Crossref PubMed Scopus (281) Google Scholar, 26Falla T.J. Karunaratne D.N. Hancock R.E.W. J. Biol. Chem. 1996; 271: 19298-19303Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). To broaden the structural variety of peptides in this study, we have added the β-sheet crab-derived peptide polyphemusin I (27Zhang L. Scott M.G. Yan H. Mayer L.D. Hancock R.E.W. Biochemistry. 2000; 39: 14504-14514Crossref PubMed Scopus (102) Google Scholar) and the cyclic bacterium-derived peptides, gramicidin S and polymyxin B (28Zhang L. Dhillon P. Yan H. Farmer S. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 3317-3321Crossref PubMed Scopus (272) Google Scholar) to this study. Our results show considerable heterogeneity in the effectiveness of these peptides in their ability to interact with model membranes. All peptides, except for gramicidin S and polymyxin B, were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis using a model 432A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) at the University of British Columbia Nucleic Acid/Protein Service. The amino acid sequence and characteristics of each peptide are shown in Table I. Gramicidin S, polymyxin B, and α-chymotrypsin and trypsin-chymotrypsin inhibitors were purchased from Sigma. The lipids PC, PG, PE, ePG, C6-NBD-PC, and DNS-PE were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). ePC and calcein were purchased from Sigma.Table IAmino acid sequence and properties of cationic antimicrobial peptides included in this studyPeptideAmino acid sequence1-aOne-letter amino acid code with the following additions: bold face indicates residues that are positively charged at neutral pH values; parentheses represent amino acids that are cyclic; O, ornothine; B, diaminobutyrate. drepresents the D-enantiomers (all other amino acids are of the L-form). Lines indicate disulphide bonds between cysteine residues.LengthNet charge6% Hydrophobic amino acidsMolecular weight1-bCharge includes the amino terminal amino group and the carboxyl terminal carboxyl group (except when amidated).Gramicidin SCyclic (LOVPFdLOVPFd)10+2801214Polymyxin BCyclized isooctanoylBTBB(BFdLBBT)10+640963IndolicidinILPWKWPWWPWRR-NH213+4771906CP11CNILKKWPWWPWRRK-NH213+6621780CP10aILAWKWAWWAWRR-NH213+4771813V681nKWKSFLKTFKSAVKTVLHTALKAISS26+6582920V25pKWKSFLRTLKSPAKTVFHTALKAISS26+6582928V8ppKWKSFLRTFKSPVRTVFHTALKPISS26+6583062Polypemusin IRRWCFRVCYRGFCYRKCR-NH218+86124581-a One-letter amino acid code with the following additions: bold face indicates residues that are positively charged at neutral pH values; parentheses represent amino acids that are cyclic; O, ornothine; B, diaminobutyrate. drepresents the D-enantiomers (all other amino acids are of the L-form). Lines indicate disulphide bonds between cysteine residues.1-b Charge includes the amino terminal amino group and the carboxyl terminal carboxyl group (except when amidated). Open table in a new tab Lipid monolayers were formed by applying the appropriate lipids dissolved in hexane or chloroform onto water contained in a circular Teflon trough (diameter = 4.5 cm, total volume of 11.5 ml). Monolayers were allowed to equilibrate until a stable surface pressure was obtained (<0.2 mN/m drift in surface pressure Δπ). A small port in the side of the trough enabled injection of reagents into the subphase without disruption of the monolayer. The subphase was gently mixed with a magnetic stir bar at 45 rpm. Surface pressure measurements were obtained by using the Whilhelmy plate method (29Mayer L.D. Nelsestuen G.L. Brockman H.L. Biochemistry. 1983; 22: 316-321Crossref PubMed Scopus (46) Google Scholar). The plate was cleaned with methanol three times and thoroughly rinsed with double-distilled water prior to each surface pressure measurement. The experiments were run at 23 °C. Symmetrically labeled unilamellar liposomes were made from an equimolar mixture of PC and PG containing 0.5 mol % C6-NBD-PC. The lipid mixture was dissolved in chloroform and was dried under a stream of nitrogen followed by 2 h of vacuum drying. The lipid film was rehydrated with TSE buffer (10 mm Tris-HCl, 150 mm NaCl, 1 mmEDTA, pH 7.5), freeze-thawed for 5 cycles and extruded 10 times through two stacked filters with a pore size of 100 nm. For inner-leaflet exclusively labeled liposomes, the symmetrically labeled unilamellar liposomes were mixed with 1 m sodium dithionite in 1m Tris-HCl, pH 7.5, and incubated for 15 min at 23 °C. The NBD groups in the outer leaflet of the bilayers were chemically quenched by the water-soluble dithionite. The liposomes were immediately separated from dithionite by gel filtration using Bio-Gel A 1.5m (Bio-Rad, Hercules, CA; 1.5 × 10 cm) at 23 °C. For making calcein-encapsulated unilamellar liposomes, a lipid film containing PC/PG (1:1) was rehydrated with 5 mm sodium HEPES, pH 7.5, containing 100 mm calcein. The liposome suspension was freeze-thawed for five cycles and extruded ten times through two stacked polycarbonate filters (100-nm pore size). The free calcein was removed by passing the liposome suspension through a Sephadex G-50 column (Amersham Pharmacia Biotech; 1.5 × 10 cm) at 23 °C and eluting with a buffer containing 20 mmsodium HEPES, 150 mm NaCl, 1 mm sodium EDTA, pH 7.5. This elution buffer was used for the calcein release experiment. Calcein-free PC/PG (1:1) unilamellar liposomes made in the same elution buffer were added to adjust final liposome concentrations in the calcein release experiment. α-Chymotrypsin-entrapped unilamellar liposomes (ePC/ePG/DNS-PE, 50:45:5) were made using 200 μm enzyme solution in buffer containing 150 mm NaCl, 20 mm HEPES, pH 7.4, according to Kobayashi et al. (23Kobayashi S. Takeshima K. Park C.B. Kim S.C. Matsuzaki K. Biochemistry. 2000; 39: 8648-8654Crossref PubMed Scopus (185) Google Scholar). Trypsin-chymotrypsin inhibitor (Sigma), at a final concentration of 200 μm, was added to the liposomes to inactivate the α-chymotrypsin outside the unilamellar liposomes. Excitation of tryptophan residues at 280 nm lead to fluorescence transfer to the dansyl group in DNS-PE leading to an emission recorded at 510 nm. A decrease in fluorescence after peptide addition implied digestion of the internalized peptide by the enzyme within the liposomes. Lipid monolayers at an air/water interface provide a simple, sensitive model for mimicking biological membranes, and many studies have shown that the monolayer technique is a powerful tool to assess membrane insertion of proteins and peptides. The primary phospholipids of Escherichia coli cells comprise a mixture of the neutral lipid phosphatidylethanolamine and the anionic lipids phosphatidylglycerol and cardiolipin, at the ratio of 78:4.7:14.4, in addition to various minor lipid species (30Hristova K. Selsted M.E. White S.H. J. Biol. Chem. 1997; 272: 24224-24233Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). We prepared monolayers from PE/ePG/CL (78:4.7:14.4) to mimic the E. coli cytoplasmic membrane and tested the ability of the peptides to interact with such monolayers. Molecules that interact only with the head groups of monolayer lipids typically induce minimal changes in surface pressure. In contrast, insertion into the hydrophobic region of the lipid monolayer can cause a significant increase in monolayer surface pressure. Thus when a protein or peptide is injected into the aqueous subphase bathing a monolayer, the degree of surface pressure change (Δπ) can be used to resolve whether peptide-membrane interactions include insertion and disturbance of the fatty acyl core of the membrane. Fig. 1 A shows the variation in surface pressure as a function of peptide concentration. A significant penetration of peptides into the hydrophobic portion of the monolayer was indicated by Δπ values of >2 mN/m (31Oishi O. Yamashita S. Nishimoto E. Lee S. Sugihara G. Ohno M. Biochemistry. 1997; 36: 4352-4359Crossref PubMed Scopus (38) Google Scholar). As observed previously for polyphemusin I (27Zhang L. Scott M.G. Yan H. Mayer L.D. Hancock R.E.W. Biochemistry. 2000; 39: 14504-14514Crossref PubMed Scopus (102) Google Scholar), all peptides induced increases in surface pressure that were a sigmoidal function of peptide concentration, a result consistent with a cooperative interaction of the peptide molecules with the monolayer. CP10A appeared to be a very effective peptide at modulating the surface pressure increase as indicated by a plateau Δπ value of ∼13 mN/m (Fig. 1 A), only surpassed by gramicidin S, which gave a maximal Δπ of 16 mN/m (28Zhang L. Dhillon P. Yan H. Farmer S. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 3317-3321Crossref PubMed Scopus (272) Google Scholar). V681n, V25p, and indolicidin appeared to be slightly less effective at modulating surface pressure increase resulting in maximal Δπ values between 10 and 12 mN/m, whereas V8pp showed modest activity with a maximal Δπ of 9 mN/m (Fig. 1 A). CP11CN showed a plateau Δπ around 7.5 mN/m (Fig. 1 A), which is nearly identical to the surface pressure increase induced by polyphemusin I (27Zhang L. Scott M.G. Yan H. Mayer L.D. Hancock R.E.W. Biochemistry. 2000; 39: 14504-14514Crossref PubMed Scopus (102) Google Scholar). These experiments were repeated at least three times and were very reproducible with differences that did not exceed 1 mN/m from experiment to experiment. The lipid avidity of each peptide was monitored by the extent of the surface pressure change upon addition of 1 μg/ml of peptide to the subphase bathing monolayers of pure PC, PE, ePG, or CL. As shown in Fig.2 B, all peptides selectively interacted with negatively charged phospholipids ePG and CL and generally had a greater effect on PG than CL monolayers. Most peptides (including polymyxin B; see Ref. 28Zhang L. Dhillon P. Yan H. Farmer S. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 3317-3321Crossref PubMed Scopus (272) Google Scholar) were not able to penetrate monolayers composed entirely of neutral lipids such as PE or PC, except indolicidin (CP10CN) and its helical variant (CP10A), which, like gramicidin S (28Zhang L. Dhillon P. Yan H. Farmer S. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 3317-3321Crossref PubMed Scopus (272) Google Scholar), displayed modest surface pressure increases of nearly 4mN/m upon binding to PE and PC monolayers (Fig.1 B). The spontaneous flip-flop rate of the fluorescent zwitterionic lipid probe, C6-NBD-PC, has been shown to be extremely small, and no measurable transfer from one monolayer to the other occurred even after 48 h of incubation in asymmetrically labeled liposomes (22Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1996; 35: 11361-11368Crossref PubMed Scopus (634) Google Scholar). Peptide-induced lipid flip-flop was measured here using unilamellar PG/PC liposomes that were asymmetrically labeled with 0.5 mol % C6-NBD-PC in the inner leaflet. Peptides were added to unilamellar liposomes, followed by the water-soluble quencher sodium dithionite (20 μl of 1m dithionite solution in 2 ml of total volume). The extent of peptide-mediated lipid flip-flop was indicated by the percentage of the NBD groups being transposed from the inner leaflet to the outer leaflet where they would then be quenched by dithionite. The peptide-induced flip-flop of the fluorescent lipid was thus recorded as a decrease of fluorescence intensity of the NBD group within the 10-min observation time period. We assessed lipid flip-flop on the time scale of 0–10 min to avoid errors because of the possible slow permeation of the quencher (dithionite) through the membrane that might occur over longer reaction times. The percent flip-flop value was defined by the following equation: percent flip-flop = 100 × (F0 −FP)/(F0 −FT), where F0,FP, and FT represent the fluorescence intensity in asymetrically labeled unilamellar liposomes without the peptide, with peptide, and with Triton X-100, respectively. Fig. 2 demonstrates the kinetics of increase in flip-flop for three peptides. The two α-helical peptides, V25p and V681n, were the most active at mediating lipid flip-flop. The flip-flop kinetics were similar for both peptides and depended on both concentration and time. At low peptide concentrations the flip-flop rate was slow, but as the peptide concentration increased the rate of increase in flip-flop also increased. V25p caused more than 95% lipid flip-flop within 300 s at 3 μg/ml (Fig.2 A), whereas V681n caused nearly 90% within 300 s at 4 μg/ml (data not shown). Conversely, the kinetics of flip-flop caused by CP10A (Fig. 2 C) and polyphemusin I (data not shown) were similar being relatively slower at any given concentration than observed for V25p and V681n. For example, CP10A resulted in only 70% flip-flop within 600 s at 5 μg/ml (Fig. 2 C), whereas polyphemusin I caused around 70% flip-flop at 3–5 μg/ml. In contrast, indolicidin, CP11CN, gramicidin S, the double-bend α-helical peptide V8pp, and polymyxin B were totally inactive at concentrations of 1–5 μg/ml at mediating lipid flip-flop when 180 μm liposomes were used (e.g. see Fig. 2 E). The extent of flip-flop after 10 min was plotted as a function of peptide concentration for eight different peptides at a range of peptide/lipid ratios, averaged over three independent experiments (Fig.3). Individual results were quite consistent, with standard errors of around 10%. In general the helical peptides V25p and V681n, the β-hairpin peptide, polyphemusin I, and the short helical indolicidin variant CP10A showed very good activity, at low peptide concentrations (peptide/lipid ratios of 1:100 to 1:400), in mediating lipid exchange between the two leaflets of a bilayer membrane (Fig. 3,A–D). This activity was clearly concentration-dependent in an apparent sigmoidal fashion. None of the other peptides, including the double-bend α-helical peptide V8pp, the extended structured peptides indolicidin and CP11CN, and the cyclic peptides gramicidin S and polymyxin B, induced any detectable lipid flip-flop at peptide/lipid ratios less than 1:100 (data not shown).Figure 3Relationship between ability of peptides to induce lipid flip-flop and membrane leakiness. The lipid composition was PC/PG (1:1). For lipid flip-flop measurements, 0.5 mol % C6-NBD-PC was included. Dotted andsolid lines represent lipid flip-flop and calcein release, respectively. Data shown are the average of three independent experiments. A–D, peptide-induced calcein release and lipid flip-flop were performed using 180 μm lipids.A, V681n; B, V25p;C, CP10A; D, polyphemusin I. E–H, similar experiments were performed using 40 μm lipids.E, gramicidin S; F, V8pp;G, CP11CN; H, indolicidin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To see whether this represented an intrinsic difference between these groups of peptides or a weaker response for the latter group, the peptide/lipid ratio was increased above 1:50, and lipid flip-flop rates were reassessed. Gramicidin S initiated lipid flip-flop at peptide/lipid ratios of 1:50 and reached a maximum (more than 90%) at peptide/lipid ratios above 1:15 (Fig. 3 E). The double-proline peptide V8pp and indolicidin induced about 50 and 40% lipid flip-flop at peptide/lipid ratios of 1:15 and 1:25, respectively (Fig. 3 F, 3H). Neither polymyxin B (data not shown) nor the indolicidin variant CP11CN (Fig.3 G) caused any detectable level of lipid flip-flop even at a peptide/lipid ratio below 1:10. To see whether lipid flip-flop was because of bilayer disruption, membrane leakage was assessed by dequenching of calcein fluorescence (which is self-quenched at the high calcein concentrations inside liposomes) after release from unilamellar PG/PC liposomes. The maximum fluorescence intensity corresponding to 100% leakage was determined by the addition of 10% (w/v) Triton X-100. The percent leakage was calculated as follows: % leakage = 100 × (FP −F0)/(FT -F0), where F0 andFP denote the fluorescence intensity before and after peptide addition, and FT represents the fluorescence intensity after addition of Triton X-100. The kinetics of calcein release is shown in Fig. 2 for three peptides using 180 μm liposomes. Both V25p (Fig.2 B) and V681n (data not shown) demonstrated similar kinetics of calcein release. At concentrations of 1–2 μg/ml, there was an immediate minor increase in peptide-mediated calcein release, but this did not increase over time. At high peptide concentrations of 4–5 μg/ml, a clearly time-dependent increase was observed (Fig. 2 B) after an initial rapid release. Neither CP10A (Fig. 2 D) nor polyphemusin I (data not shown) were effective at causing calcein release from unilamellar liposomes, and only at 5 μg/ml was any more than 20% calcein release observed (Fig. 2 D). Indolicidin, CP11CN, V8pp, gramicidin S, and polymyxin B were unable to induce calcein release at the tested concentrations using 180 μm liposomes (e.g. see Fig. 2 F). These results were summarized for eight peptides at several peptide/lipid ratios for three independent experiments (Fig. 3). Calcein release induced by most peptides demonstrated a sigmoidal dependence on peptide concentration. There was little calcein release at low peptide concentrations, indicating that there was a threshold peptide concentration for stimulating calcein release, although the threshold concentrations differed among the peptides. Both V681n and V25p induced significant membrane damage at peptide/lipid ratios above 1:260 (Fig. 3, A andB). Nevertheless, for V681n and V25p, the concentration resulting in 50% calcein release was 2.5- and 5-fold higher than the concentration that

The solar-driven water splitting process is highly attractive for alternative energy utilization, while developing efficient, earth-abundant, bifunctional catalysts for both oxygen evolution reaction and hydrogen evolution reaction has remained as a major challenge. Herein, we develop an ordered CoMnO@CN superlattice structure as an efficient bifunctional water-splitting electrocatalyst, in which uniform Co-Mn oxide (CoMnO) nanoparticles are coated with a thin, continuous nitrogen-doped carbon (CN) framework. The CoMnO nanoparticles enable optimized OER activity with effective electronic structure configuration, and the CN framework serves as an excellent HER catalyst. Importantly, the ordered superlattice structure is beneficial for enhanced reactive sites, efficient charge transfer, and structural stability. This bifunctional superlattice catalyst manifests optimized current densities and electrochemical stability in overall water splitting, outperforming most of the previously reported single- or bifunctional electrocatalysts. Combining with a silicon photovoltaic cell, this CoMnO@CN superlattice bifunctional catalyst enables unassisted solar water splitting continuously for ∼5 days with a solar-to-hydrogen conversion efficiency of ∼8.0%. Our discovery suggests that these transition metal oxide-based superlattices may serve as a unique structure modality for efficient bifunctional water splitting electrocatalysts with scale-up potentials.

Ag/CeO2 catalysts employing CeO2 nanocubes (c-CeO2) and nanorods (r-CeO2) as the support were prepared by conventional incipient wetness impregnation followed by calcination at 500 °C in air. Their structures have been characterized in detail and their catalytic activities in CO oxidation have also been tested. c-CeO2 and r-CeO2 nanocrystals exhibit different concentrations and structures of oxygen vacancies. The silver-r-CeO2 interaction is stronger than the silver-c-CeO2 interaction. Fine Ag nanoparticles form in 1%-Ag/c-CeO2 and grow in size in 3%-Ag/c-CeO2; however, positively charged Agn+ clusters dominate in 1%-Ag/r-CeO2, and fine Ag nanoparticles dominate in 3%-Ag/r-CeO2. Supported Ag nanoparticles are much more capable of creating oxygen vacancies in CeO2 than supported positively charged Agn+ clusters. More oxygen vacancies form in Ag/c-CeO2 than in Ag/r-CeO2. The average charge density of oxygen vacancies and the ratio between large oxygen vacancy clusters and small vacancies in CeO2 nanocrystals are enhanced when loaded with positively charged Agn+ clusters but reduced when loaded with Ag nanoparticles. Ag nanoparticles greatly promote the reduction and catalytic activity in CO oxidation of CeO2 nanocrystals but positively charged Agn+ clusters do not. These results demonstrate the concept that the interplay between oxygen vacancies and Ag–CeO2 interaction controls the structures of silver and CeO2 in Ag/CeO2 catalysts and thus their surface reactivity and catalytic activity, deepening the fundamental understanding of metal/CeO2 catalysts. These results also reveal that the interplay between oxygen vacancies and Ag–CeO2 interaction in Ag/CeO2 catalysts depends on the shape of CeO2 support, opening up a new strategy for the design of efficient and economic metal/CeO2 catalysts by engineering the shape of CeO2 support.

Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electrocatalysis. Conducting polymer-based electrochemical DNA sensors have shown applicability in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity. This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immobilization and detection methodologies are presented. Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed.

A novel boronic acid functionalized mesoporous silica, which holds the attractive features of high surface area and large accessible porosity, was developed to enrich glycopeptides. This is the first time that mesoporous material has been introduced into glycoproteome. In comparison to direct (traditional) analysis, this novel method enabled 2 orders of magnitude improvement in the detection limit of glycopeptides. The unbiased nature of organo-boronic acid groups also made this method applicable to all kinds of glycopeptides regardless of their sizes, structures, and hydrophilicities.

Metal–organic frameworks (MOFs) with open metal sites enrich the population of O2 in the pores significantly and assist the Li–O2 reaction when employed as a cell electrode material. A primary capacity of 9420 mA h g−1 is achieved in a cell with Mn-MOF-74; more than four times higher than the value obtained in a cell without an MOF.

Uniform oxide deposition on graphene to form a sandwich-like configuration is a well-known challenge mainly due to their large lattice mismatches and poor affinities. Herein, we report a general strategy to synthesize uniform mesoporous TiO2/graphene/mesoporous TiO2 sandwich-like nanosheets (denoted as G@mTiO2), which cannot be achieved by conventional one-pot synthetic methods. We show that by rational control of hydrolysis and condensation of Ti precursors in a slow way, GO sheets can be conformably coated by amorphous TiO2 shells, which then can be facilely transformed into the well-defined G@mTiO2 nanosheets by annealing. This amorphous-to-crystalline strategy conveniently allows bypassing strain fields that would inevitably arise if direct growth of mesoporous anatase shells on graphene. As distinct from the most common structures of graphene-based composites (mixed, wrapped, or anchored models), the resultant materials display a uniform sandwich-like configuration: few-layer graphene conformably encapsulated by mesoporous TiO2 shells. This new G@mTiO2 nanosheet exhibits ultrathin nature (∼34 nm), small size and high crystalline nanocrystals (∼6 nm), high surface areas (∼252 m2/g) and uniform mesopores (∼3.4 nm). We further show that the thickness of mesoporous TiO2 shells can be facilely adjusted as desired by controlling the ammonia content, and this facile strategy can be easily extended to design other oxide/graphene/oxide sandwich-like materials. More importantly, we showcase the benefits of the resultant G@mTiO2 nanosheets as anodes in lithium ion batteries: they deliver an extra high capacity, an excellent high-rate capability, and long cycle life.

A simple DNA sensing platform was developed based on the fluorescence resonance energy transfer (FRET) between blue-luminescent CdTe QDs and dye-labeled ssDNA. A cationic polymer acts as a "bridge" to bring the donor to acceptor together and achieves the high FRET efficiency. The hybridization event was recognized by the different FRET efficiencies due to different interaction abilities of single-stranded and double-stranded DNA with the polymer. This platform provides a homogeneous DNA assay that takes advantage of the fluorescence detection method, but with minimal DNA modification.

NiS₂–MoS₂ interlaced nanoflake-nanowires were synthesized by the thermal conversion of NiMoO₄ nanowire precursors as attractive hydrogen evolution catalysts in basic solutions.

This study investigated the occurrence of 43 emerging contaminants including 9 endocrine-disrupting chemicals and 34 pharmaceuticals in three sites in Hebei Province, north China. Each site has a wastewater irrigated plot and a separate groundwater irrigated plot for comparison purpose. The results showed that the concentrations of the target compounds in the wastewater irrigated soils were in most cases higher than those in the groundwater irrigated soils. Among the 43 target compounds, nine compounds bisphenol-A, triclocarban, triclosan, 4-nonylphenol, salicylic acid, oxytetracycline, tetracycline, trimethoprim and primidone were detected at least once in the soils. Preliminary environmental risk assessment showed that triclocarban might pose high risks to terrestrial organisms while the other detected compounds posed minimal risks. Irrigation with wastewater could lead to presence or accumulation of some emerging contaminants to some extent in irrigated soils.

A hierarchical architecture of Ti3C2/Fe3O4/PANI composite as a high-efficiency microwave absorber was synthesized using HF etching, coprecipitation and in-situ polymerization route. Layered Ti3C2 was used as a matrix material with high surface area and a network offering more paths of electron transfer. The adding of PANI and Fe3O4 can generate an outstanding microwave absorption property because of enhanced interfacial polarization, strong attenuation loss and excellent impedance matching. The hierarchical Ti3C2/Fe3O4/PANI ternary composite shows a strongest reflection loss (RL) of −40.3 dB at 15.3 GHz, which is higher than those of Fe3O4 and Ti3C2/Fe3O4. Furthermore, it also displays broad absorption frequency band, possessing the efficient absorption bandwidth (<−10 dB) of 5.2 GHz (from 12.8 to18 GHz) at only 1.9 mm. The improving microwave absorption performances may be ascribed to hierarchical structure and synergistic effect of combining Ti3C2, Fe3O4 and PANI. The results of this work provide a new idea of utilizing Ti3C2 matrix to prepare 3D hierarchical composite for the applications of MXene family in microwave absorption fields.

Accurate chromosome segregation depends on the proper attachment of sister kinetochores to microtubules emanating from opposite spindle poles. Merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. Despite correction mechanisms, merotelically attached kinetochores can persist until anaphase, causing chromatids to lag on the mitotic spindle and hindering their timely segregation. Recent studies showing that merotelic kinetochore attachment represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells have underlined the importance of studying merotely. Here, we highlight recent progress in our understanding of how cells prevent and correct merotelic kinetochore attachments.

Most of traditional Chinese medicine substances come from herbal plants. The medicinal quality of herbal plants varies with the locations of cultivation, the parts of the herb collected, the season of the herb collected, and the herb processing method. Polysaccharides are major components of the herb plants and their biosynthesis is partly controlled by the genes but mostly influenced by the availability of the nutrition and determined by the various environmental factors. In recent decades, polysaccharides isolated from different kinds of Chinese herbs have received much attention due to their important biological activities, such as anti-tumor, anti-oxidant, anti-diabetic, radiation protecting, antiviral, hypolipidemic, and immunomodulatory activities. Interestingly, different batches of the same herb can obtain different polysaccharide fractions with subtle differences in molecular weight, monosaccharide compositions, glycosidic linkages, and biological functions. Even with these variations, a large number of bioactive polysaccharides from different kinds of traditional Chinese herbs have been purified, characterized, and reported. This review provides a comprehensive summary of the latest polysaccharide extraction methods and the strategies used for monosaccharide compositional analysis plus polysaccharide structural characterization. Most importantly, the reported chemical characteristics and biological activities of the polysaccharides from the famous traditional Chinese herbs including Astragalus membranaceus, Ginseng, Lycium barbarum, Angelica sinensis, Cordyceps sinensis, and Ophiopogon japonicus will be reviewed and discussed. The published studies provide evidence that polysaccharides from traditional Chinese herbs play an important role in their medical applications, which forms the basis for future research, development, and application of these polysaccharides as functional foods and therapeutics in modern medicine.

Major depressive disorder (MDD) is a common emotional cognitive disorder that seriously affects people's physical and mental health and their quality of life. Due to its clinical and etiological heterogeneity, the molecular mechanisms underpinning MDD are complex and they are not fully understood. In addition, the effects of traditional drug therapy are not ideal. However, postmortem and animal studies have shown that overactivated microglia can inhibit neurogenesis in the hippocampus and induce depressive-like behaviors. Nonetheless, the molecular mechanisms by which microglia regulate nerve regeneration and determine depressive-like behaviors remain unclear. As the immune cells of the central nervous system (CNS), microglia could influence neurogenesis through the M1 and M2 subtypes, and these may promote depressive-like behaviors. Microglia may be divided into four main states or phenotypes. Under stress, microglial cells are induced into the M1 type, releasing inflammatory factors and causing neuroinflammatory responses. After the inflammation fades away, microglia shift into the alternative activated M2 phenotypes that play a role in neuroprotection. These activated M2 subtypes consist of M2a, M2b and M2c and their functions are different in the CNS. In this article, we mainly introduce the relationship between microglia and MDD. Importantly, this article elucidates a plausible mechanism by which microglia regulate inflammation and neurogenesis in ameliorating MDD. This could provide a reliable basis for the treatment of MDD in the future.

Abstract Nitrogen‐doped carbon structures have recently been demonstrated as a promising candidate for electrocatalytic CO 2 reduction, while in the meantime the pyridinic and graphitic nitrogen atoms also present high activities for electroreduction of water. Here, an etching strategy that uses hot water steam to preferentially bind to pyridinic and graphitic nitrogen atoms and subsequently etch them in carbon frameworks is reported. As a result, pyrrolic nitrogen atoms with low water affinity are retained after the steam etching, with a much increased level of among all nitrogen species from 22.1 to 55.9%. The steam‐etched nitrogen‐doped carbon catalyst enables excellent electrocatalytic CO 2 reduction performance but low hydrogen evolution reaction activity, suggesting a new approach for tuning electrocatalyst activity.

Systemic lupus erythematosus (SLE) patients are at high risk for depression and anxiety. However, the estimated prevalence of these disorders varies substantially between studies. This systematic review aimed to establish pooled prevalence levels of depression and anxiety among adult SLE patients. We systematically reviewed databases including PubMed, Embase, PsycINFO, and the Cochrane database library from their inception to August 2016. Studies presenting data on depression and/or anxiety in adult SLE patients and having a sample size of at least 60 patients were included. A random-effect meta-analysis was conducted on all eligible data. A total of 59 identified studies matched the inclusion criteria, reporting on a total of 10828 adult SLE patients. Thirty five and thirteen methods of defining depression and anxiety were reported, respectively. Meta-analyses revealed that the prevalence of major depression and anxiety were 24% (95% CI, 16%-31%, I2 = 95.2%) and 37% (95% CI, 12%–63%, I2 = 98.3%) according to clinical interviews. Prevalence estimates of depression were 30% (95% CI, 22%–38%, I2 = 91.6%) for the Hospital Anxiety and Depression Scale with thresholds of 8 and 39% (95% CI, 29%–49%, I2 = 88.2%) for the 21-Item Beck Depression Inventory with thresholds of 14, respectively. The main influence on depression prevalence was the publication years of the studies. In addition, the corresponding pooled prevalence was 40% (95% CI, 30%–49%, I2 = 93.0%) for anxiety according to the Hospital Anxiety and Depression Scale with a cutoff of 8 or more. The prevalence of depression and anxiety was high in adult SLE patients. It indicated that rheumatologists should screen for depression and anxiety in their patients, and referred them to mental health providers in order to identify effective strategies for preventing and treating depression and anxiety among adult SLE patients. Current Meta-analysis PROSPERO Registration Number: CRD 42016044125 . Registered 4 August 2016.

Self-assembly of inorganic nanoparticles into ordered structures is of interest in both science and technology because it is expected to generate new properties through collective behavior; however, such nanoparticle assemblies with characteristics distinct from those of individual building blocks are rare. Herein we use atomically precise Au clusters to make ordered assemblies with emerging optical activity. Chiral Au clusters with strong circular dichroism (CD) but free of circularly polarized luminescence (CPL) are synthesized and organized into uniform body-centered cubic (BCC) packing nanocubes. Once the ordered structure is formed, the CD intensity is significantly enhanced and a remarkable CPL response appears. Both experiment and theory calculation disclose that the CPL originates from restricted intramolecular rotation and the ordered stacking of the chiral stabilizers, which are fastened in the crystalline lattices.

A graphdiyne nanosheet/Pt nanoparticle composite shows outstanding catalytic activity for the reaction of I3−/I− redox pairs, i.e., the best among rGO/Pt nanoparticle composites, Pt nanoparticles and Pt foils. This is ascribed to enhanced electron transfer resulting from the easier chemical interaction between Pt and the triple bond of graphdiyne.

Several studies have shown that tRNAs can be enzymatically cleaved to generate distinct classes of tRNA-derived fragments (tRF). Here, we report that tRF/miR-1280, a 17-bp fragment derived from tRNALeu and pre-miRNA, influences Notch signaling pathways that support the function of cancer stem-like cells (CSC) in colorectal cancer progression. tRF/miR-1280 expression was decreased in human specimens of colorectal cancer. Ectopic expression of tRF/miR-1280 reduced cell proliferation and colony formation, whereas its suppression reversed these effects. Mechanistic investigations implicated the Notch ligand JAG2 as a direct target of tRF/miR-1280 binding through which it reduced tumor formation and metastasis. Notably, tRF/miR-1280-mediated inactivation of Notch signaling suppressed CSC phenotypes, including by direct transcriptional repression of the Gata1/3 and miR-200b genes. These results were consistent with findings of decreased levels of miR-200b and elevated levels of JAG2, Gata1, Gata3, Zeb1, and Suz12 in colorectal cancer tissue specimens. Taken together, our results established that tRF/miR-1280 suppresses colorectal cancer growth and metastasis by repressing Notch signaling pathways that support CSC phenotypes. Furthermore, they provide evidence that functionally active miRNA can be derived from tRNA, offering potential biomarker and therapeutic uses. Cancer Res; 77(12); 3194-206. ©2017 AACR.

Exosomes have emerged as a promising drug carrier with low immunogenicity, high biocompatibility and delivery efficiency. Here in, we isolated exosomes from A33-positive LIM1215 cells (A33-Exo) and loaded them with doxorubicin (Dox). Furthermore, we coated surface-carboxyl superparamagnetic iron oxide nanoparticles (US) with A33 antibodies (A33Ab-US), expecting that these A33 antibodies on the surface of the nanoparticles could bind to A33-positive exosomes and form a complex (A33Ab-US-Exo/Dox) to target A33-positive colon cancer cells. The results showed that A33Ab-US-Exo/Dox had good binding affinity and antiproliferative effect in LIM1215 cells, as shown by increased uptake of the complex. In vivo study showed that A33Ab-US-Exo/Dox had an excellent tumor targeting ability, and was able to inhibit tumor growth and prolong the survival of the mice with reduced cardiotoxicity. In summary, exosomes functionalized by targeting ligands through coating with high-density antibodies may prove to be a novel delivery system for targeted drugs against human cancers.

Accumulating evidence indicates that circular RNAs (circRNAs) are vital regulators of various biological functions involved in the progression of multiple cancers. Circular F-box and WD repeat domain containing 7 (circFBXW7) (hsa_circ_0001451) has been reported to act as a tumor suppressor by encoding a novel protein in glioma; however, its functions and mechanisms in triple-negative breast cancer (TNBC) remain elusive. In the current study, we validated by qRT-PCR that circFBXW7 was downregulated in TNBC cell lines and found that low expression of circFBXW7 was associated with poorer clinical outcomes. circFBXW7 expression was negatively correlated with tumor size and lymph node metastasis, and it was an independent prognostic factor for TNBC patients. We performed cell proliferation, colony formation, transwell, wound-healing, and mouse xenograft assays to confirm the functions of circFBXW7. Overexpression of circFBXW7 obviously inhibited cell proliferation, migration, and tumor growth in both <i>in vitro</i> and <i>in vivo</i> assays. Luciferase reporter assays and RNA immunoprecipitation assays revealed that circFBXW7 serves as a sponge of miR-197-3p and suppresses TNBC growth and metastasis by upregulating FBXW7 expression. In addition, the FBXW7-185aa protein encoded by circFBXW7 inhibited the proliferation and migration abilities of TNBC cells by increasing the abundance of FBXW7 and inducing c-Myc degradation. In summary, our research demonstrated that circFBXW7 sponges miR-197-3p and encodes the FBXW7-185aa protein to suppress TNBC progression through upregulating FBXW7 expression. Thus, circFBXW7 may act as a therapeutic target and prognostic biomarker for TNBC.

Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P < 3.65 × 10(-5)). Eight independent SNPs overlapped across Han Chinese, European and U.S. populations, and APOE and 5q33.3 were replicated as longevity loci. Integrated analysis indicates four pathways (starch, sucrose and xenobiotic metabolism; immune response and inflammation; MAPK; calcium signaling) highly associated with longevity (P ≤ 0.006) in Han Chinese. The association with longevity of three of these four pathways (MAPK; immunity; calcium signaling) is supported by findings in other human cohorts. Our novel finding on the association of starch, sucrose and xenobiotic metabolism pathway with longevity is consistent with the previous results from Drosophilia. This study suggests protective mechanisms including immunity and nutrient metabolism and their interactions with environmental stress play key roles in human longevity.

Extracellular matrices (ECMs) are central to the advent of multicellular life, and their mechanical properties are modulated by and impinge on intracellular signaling pathways that regulate vital cellular functions. High spatial-resolution mapping of mechanical properties in live cells is, however, extremely challenging. Thus, our understanding of how signaling pathways process physiological signals to generate appropriate mechanical responses is limited. We introduce fluorescence emission-Brillouin scattering imaging (FBi), a method for the parallel and all-optical measurements of mechanical properties and fluorescence at the submicrometer scale in living organisms. Using FBi, we showed that changes in cellular hydrostatic pressure and cytoplasm viscoelasticity modulate the mechanical signatures of plant ECMs. We further established that the measured "stiffness" of plant ECMs is symmetrically patterned in hypocotyl cells undergoing directional growth. Finally, application of this method to Arabidopsis thaliana with photoreceptor mutants revealed that red and far-red light signals are essential modulators of ECM viscoelasticity. By mapping the viscoelastic signatures of a complex ECM, we provide proof of principle for the organism-wide applicability of FBi for measuring the mechanical outputs of intracellular signaling pathways. As such, our work has implications for investigations of mechanosignaling pathways and developmental biology.

Inflammasome activation plays key roles in host defense, but also contributes to the pathogenesis of auto-inflammatory, and neurodegenerative diseases. As autophagy is connected with both the innate and adaptive immune systems, autophagic dysfunction is also closely related to inflammation, infection, and neurodegeneration. Here we identify that lincRNA-Cox2, previously known as a mediator of both the activation and repression of immune genes expression in innate immune cells, could bind NF-κB p65 and promote its nuclear translocation and transcription, modulating the expression of inflammasome sensor NLRP3 and adaptor ASC. Knockdown of lincRNA-Cox2 inhibited the inflammasome activation and prevented the lincRNA-Cox2-triggered caspase-1 activation, leading to decreased IL-1β secretion and weakened TIR-domain-containing adapter-inducing interferon-β (TRIF) cleavage, thereby enhancing TRIF-mediated autophagy. Elucidation of the link between lincRNA-Cox2 and the inflammasome-autophagy crosstalk in macrophage and microglia reveals a role for lncRNAs in activation of NLRP3 inflammasome and autophagy, and provides new opportunities for therapeutic intervention in neuroinflammation-dependent diseases.

In this paper, a facilely prepared electrochemiluminescence (ECL) biosensor was developed for Escherichia coli O157:H7 quantitative detection based on a polydopamine (PDA) surface imprinted polymer (SIP) and nitrogen-doped graphene quantum dots (N-GQDs). N-GQDs with a high quantum yield of 43.2% were synthesized. The uniform PDA SIP film for E. coli O157:H7 was established successfully with a facile route. The dopamine and target bacteria were electropolymerized directly on the electrode. After removal of the E. coli O157:H7 template, the established PDA SIP can selectively recognize E. coli O157:H7. Accordingly, E. coli O157:H7 polyclonal antibody (pAb) was labeled with N-GQDs. The bioconjugation of SIP–E. coli O157:H7/pAb-N-GQDs can generate intensive ECL irradiation with K2S2O8. As a result, E. coli O157:H7 was detected with the ECL sensing system. Under optimal conditions, the linear relationships between the ECL intensity and E. coli O157:H7 concentration were obtained from 101 colony-forming units (CFU) mL–1 to 107 CFU mL–1 with a limit of detection of 8 CFU mL–1. The biosensor based on this SIP film was applied in water sample detection successfully. The N-GQD-based ECL analytical method for E. coli O157:H7 was reported for the first time. The sensing system had high selectivity to the target analyte, provided new opportunities for use, and increased the rate of disease diagnosis and treatment and the prevention of pathogens.

Increasing studies has found that circular RNAs (circRNAs) play vital roles in cancer progression. But the expression profile and function of circRNAs in triple-negative breast cancer (TNBC) are unclear.We used a circRNA microarray to explore the circRNA expression profile of TNBC. The expression of the top upregulated circRNA, circKIF4A, was confirmed by qRT-PCR in breast cancer cell lines and tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of circKIF4A on TNBC. A series of experiments was performed to explore the functions of circKIF4A in TNBC progression, such as cell proliferation and migration. We investigated the regulatory effect of circKIF4A on miRNA and its target genes to explore the potential regulatory mechanisms of circKIF4A in TNBC.qRT-PCR analyses verified that circKIF4A was significantly upregulated and positively associated with poorer survival of TNBC. The inhibition of circKIF4A suppressed cell proliferation and migration in TNBC. Luciferase reporter assay and RNA immunoprecipitation assay revealed that circKIF4A and KIF4A could bind to miR-375 and that circKIF4A regulated the expression of KIF4A via sponging miR-375.The circKIF4A-miR-375-KIF4A axis regulates TNBC progression via the competitive endogenous RNA (ceRNA) mechanism. circKIF4A may therefore serve as a prognostic biomarker and therapeutic target for TNBC.

The graphene oxide (GO) functionalized by Fe3O4@SiO2@CS-TETA nanoparticles, Fe3O4@SiO2@CS-TETA-GO, was firstly fabricated in a mild way as a novel adsorbent for the removal of Cu(II) ions and methylene blue (MB) from aqueous solutions. The magnetic composites showed a good dispersity in water and can be conveniently collected for reuse through magnetic separation due to its excellent magnetism. When the Fe3O4@SiO2@CS- TETA-GO was used as an absorbent for the absorption of MB and Cu(II), the adsorption kinetics and isotherms data well fitted the pseudo-second-order model and the Langmuir model, respectively. Under the optimized pH and initial concentration, the maximum adsorption capacity was about 529.1 mg g−1 for MB in 20 min and 324.7 mg g−1 for Cu(II) in 16 min, respectively, exhibiting a better adsorption performance than other GO-based adsorbents reported recently. More importantly, the synthesized adsorbent could be effectively regenerated and repeatedly utilized without significant capacity loss after six times cycles. All the results demonstrated that Fe3O4@SiO2@CS-TETA-GO could be used as an excellent adsorbent for the adsorption of Cu(II) and MB in many fields.

Wastewater indicator is a useful tool for evaluating the wastewater impact on natural water, but there is little information about the suitability of wastewater indicators for different regions. This study aimed to select suitable wastewater indicators in the Pearl River Delta region, south China by screening a range of wastewater related organic compounds. The screening campaign was carried out by investigating the occurrence and removal efficiencies of 93 pharmaceuticals and personal care products (PPCPs) and 5 artificial sweeteners (ASs) in nine wastewater treatment plants (WWTPs) located in the region, and the occurrence of these target compounds in the contaminated and clean surface water of the Pearl River. An ideal wastewater indicator should be hydrophilic, source-specific for domestic wastewater, ubiquitous in contaminated surface water with detection frequency (DF) > 80% and absent in background water samples. For liable indicators, high removal rates (> 90%) should be observed in WWTPs and they should be detected in all the influent samples at concentrations fifty times higher than their limits of quantification. For conservative indicators, low removal rates (< 50%) should be observed in WWTPs and they should be detected in all the effluent samples at concentrations fifty times higher than their limits of quantification. Based on the above criteria, sucralose and fluconazole were selected as conservative indicators in the region, while cyclamate, saccharin, methyl paraben, ethyl paraben, propyl paraben, paracetamol, salicylic acid and caffeine were selected as liable indicators.

Hippocampal microglia mediate stress resilience.

The cold metal transfer (CMT) based wire-arc additive manufacturing (WAAM) technology has been widely recognized as a suitable method for fabricating large-sized aluminum alloy components. However, the poor mechanical properties of the as-deposited aluminum alloys prevent their wide application in the aerospace industry. In this paper, three categories of samples with different interlayer deformation strains were fabricated by WAAM. These samples were further investigated to evaluate the effects of interlayer deformation on the mechanical properties, microstructural evolution, and the underlying strengthening mechanism. The grain size distribution and internal sub-microstructure were characterized by electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). As compared to the as-deposited samples, the yield strength and ultimate tensile strength of the 50.8% deformed sample increased from 148.4 to 240.9 MPa and from 288.6 to 334.6 MPa, respectively. The microstructure of the samples with interlayer hammering exhibited highly refined grain, which is a combined result of deformation and subsequent intrinsic in-situ heat treatment induced by the next deposition layer. The recrystallized grains can be further deformed with subsequent hammering, which leads to an increase in dislocation density and contributes to an increase in ultimate tensile strength of the additively manufactured 2319 aluminum alloys with interlayer hammering.

The electrochemical reduction of carbon dioxide and nitrite ions into value-added chemicals represents one of the most promising approaches to relieve the greenhouse gases, while a critical challenge is to search for a highly effective catalyst with low energy input and high conversion selectivity. In this work, we demonstrated low-valence Cu doped, oxygen vacancy-rich anatase TiO2 (Cu-TiO2) nanotubes as a synergetic catalyst for electrochemical co-reduction of both CO2 and NO2–. The incorporation of Cu dopants in anatase TiO2 facilitated to form abundant oxygen vacancies and bi-Ti3+ defect sites, which allowed for efficient nitrite adsorption and activation. The low-valence Cu dopants also served as effective catalytic centers to reduce CO2 into CO* adsorbate. The close proximity of CO* and NH2* intermediates was beneficial for the subsequent cooperative tandem reaction to form urea via the CN coupling. This oxygen vacancy-rich Cu-TiO2 electrocatalyst enabled excellent urea production rate (20.8 μmol⋅h−1) and corresponding Faradaic efficiency (43.1%) at a low overpotential of –0.4 V versus reversible hydrogen electrode, substantially superior than those of undoped TiO2, thus suggesting an exciting approach for cooperative CO2 and nitrogen fixation.

Flexible pressure sensors have promising applications in wearable electronic devices. However, fabricating flexible pressure sensors with wide linear range and high sensitivity remain a great challenge. Herein, a micro-nano hybrid conductive elastomer film based on carbon materials with arched micro-patterns array on surface (P-HCF) is developed to show expected sensing properties through a sustainable route. The 1D carbon fibers (CFs) and 0D carbon nanoparticles (CNPs) were incorporated into polydimethylsiloxane (PDMS) matrix to construct a 3D conductive network consisting of physical contact and tunneling effect among carbon materials to improve the sensing range and sensitivity. The arched micro-patterns of the P-HCF, which is designed mimicking the human fingerprints, influences the pressure distribution inside the material, giving rise to a linear sensitivity over the whole sensing range. Finite element analysis (FEA) method is investigated to simulate and analyze the compression process. The P-HCF sensor exhibits both a high sensitivity of 26.6 kPa−1 and an exceptionally wide linear range of 20 Pa − 600 kPa. The devices were demonstrated in monitoring artery pulses, assisting in diagnosing Parkinson’s disease, and analyzing gait for healthcare. Furthermore, the sensors are integrated into complex devices to realize pressure distribution detection, controlling manipulator, and operating PC games. The attainment of excellent pressure sensing performance of the P-HCF, potentially initiates vast applications in health monitoring and human-machine interfaces.

Sulfonamide antibiotic contamination promotes the generation of resistance genes, whereas its removal remains a great challenge due to the lack of understanding in its removal micro-mechanism and efficient adsorbents. In this study, the interfacial interaction mechanism associated with the adsorption of sulfamethazine (SMT) on mesoporous cellulose biochar (MCB) adsorbents were studied, as well as the influences of pH value, cations and humic acid (HA) on this interfacial interaction mechanism. A π-electron interaction, not electrostatic interactions, is the main interfacial interaction mechanism. A thermodynamic and kinetic study revealed that the monolayer adsorption occurred and was dominated by chemisorption. Density functional theory (DFT) and frontier orbital theory (FOT) simulations were conducted to further explore the interfacial interaction micro-mechanism at the molecular and electronic levels. The adsorption equilibrium configurations confirmed that SMT was adsorbed onto MCB in the V-shape configuration, mainly through π-π EDA interactions. DFT simulations showed that increasing the π electron density of the biochar favored its adsorption of SMT and that the pyrrole groups might be the most effective functionalities for this adsorption. The above findings could provide significant reference value for the design and preparation of efficient sulfonamide antibiotic biochar adsorbents.

Abstract TiO 2 is a potential anode material for lithium‐ion batteries due to its high rate capability and high safety. Here, a controllable synthesis for hollow nanostructured TiO 2 , with heterostructured shells of TiO 2 (B) and anatase phases, is presented for the first time, by using a sequential templating approach. The hollow nanostructures can be easily controlled to produce core–shell and double‐shelled materials with different compositional ratios of anatase to TiO 2 (B) by tuning the synthetic conditions. When used as the anode materials for lithium‐ion batteries, a specific discharge capacity of 215.4 mAh g −1 for the double‐shelled anatase/TiO 2 (B) hollow microspheres is achieved at a current rate of 1 C (335 mA g −1 ) for the 100th cycle and shows high specific discharge capacities of 141.6 and 125.7 mAh g −1 at the high rates of 10 and 20 C over 1000 cycles. These results are due to the unique stable hollow multishelled structure, which has a high specific surface area, as well as the interface between the heterostructured anatase/TiO 2 (B) phases contributing a substantial number of lithium‐ion storage sites.

Heavy metal contamination directly threatened human life and health. In this work, a novel carboxyl, amide, carbonyl sulfide and secondary amino group grafted cellulose derivative adsorbent (modified-cellulose) was prepared in an attempt to remove heavy metal Cd2+. The XRD, FTIR, 13C NMR and XPS results showed that the carboxyl, amide, carbonyl sulfide and secondary amino group were grafted onto the cellulose backbone successfully. Effects of various factors on the adsorption performance were investigated as well as the adsorption mechanism. The Cd2+ adsorption capacity of modified-cellulose was pretty good, up to 401.1 mg/g and with 3 times enhancement. The adsorption process was spontaneous, well described by the Freundlich model (R2 = 0.994), confirmed to the pseudo-second-order model (R2 > 0.997), and mainly controlled by chemisorption. The density functional theory (DFT) calculations indicating that the Cd2+ binding ability of multi-functional groups modified cellulose was stronger than that of single-functional group modified cellulose. The preferential adsorption sites were analyzed based on the frontier orbital theory (FOT), and they were concentrated on the secondary amino groups and carbonyl sulfides. It is foreseeable that the as-prepared modified-cellulose adsorbent has great potential in heavy metal cadmium removal, and our conclusions could provide significant theoretical guidance in the due bioresource utilization.

Chiral fluorescent materials with fluorescent nanoparticles assembled into a chiral structure represent a grand challenge. Here, we report self-assembled emissive needle-like nanostructures through decorating cellulose nanocrystals (CNCs) with carbon quantum dots (CQDs). This assembly is facilitated by the heterogeneous amphiphilic interactions between natural and synthetic components. These emissive nanostructures can self-organize into chiral nematic solid-state materials with enhanced mechanical performance. The chiral CQD/CNC films demonstrate an intense iridescent appearance superimposed with enhanced luminescence that is significantly higher than that for CQD films and other reported CQD/CNC films. A characteristic fluorescent fingerprint signature is observed in the CQD/CNC film, proving the well-defined chiral organization of fluorescent nanostructures. The chiral organization of CQDs enables the solid CQD/CNC film to form a right-hand chiral fluorescence with an asymmetric factor of −0.2. Additionally, we developed chemical 2D printing and soft lithography patterning techniques to fabricate the freestanding chiral fluorescent patterns that combines mechanical intergrity and chiral nematic structure with light diffraction and emission.

The anti-inflammatory effects of phytochemicals, bioactive components from plants having health benefits, have been heavily investigated in the last several decades. However, the gap between the high dosage demands (μM) of phytochemicals in vitro studies and the low bioavailability (nM) of most phytochemicals after consuming relevant foods/supplements in humans undermines the application of these phytochemicals in the prevention of chronic inflammation and its related chronic diseases in humans. One of the approaches to bridging this gap is to combine two or more phytochemicals/foods to synergistically prevent chronic inflammation. While increasing combinations of phytochemicals on anti-inflammation studies have been reported, there is no report dedicating why combining two or more phytochemicals synergistically attenuates chronic inflammation. In the present review, we summarized different types of combinations exerting synergistic anti-inflammatory effects such as the combination of phytochemicals from the same foods, and the combination of phytochemicals from different foods/plants. Particularly, we proposed five mechanisms including enhancing the bioavailability of phytochemicals, increasing antioxidant capacity, interacting with gut microbiome and targeting same and different signaling pathways, to understand how the combination of phytochemicals exerts synergistic anti-inflammatory effects in cells, animals, and humans. This review provides clues to boost more studies to combine several phytochemicals/foods to reduce chronic inflammation and prevent chronic diseases in humans.

Food-grade high internal phase Pickering emulsions (HIPPEs) stabilized by protein-based particles have received widespread attention because of their potential applications in the food industry. Herein, HIPPEs stabilized by sea bass protein (SBP) microgel particles were prepared using a simple one-step method. Its internal phase volume fraction was as high as 88% oil-in-water emulsion. The impact of SBP microgel particles concentration on the physical and chemical properties of HIPPEs was investigated. The SBP microgel particles improved the environmental stability of HIPPEs. Confocal laser scanning microscope (CLSM) and cryo-scanning electron microscope (cryo-SEM) images showed a three-dimensional network structure formed around oil droplets through SBP microgel particles. The average particles size of the HIPPEs droplets decreased with the increased concentration of SBP microgel particles. In rheological analysis, as the concentration of SBP microgel particles increased, HIPPEs showed higher viscoelasticity, excellent recovery, and thixotropy, which further proved the potential application of HIPPEs in 3D printing. The physical and chemical stability of astaxanthin was improved after encapsulation of HIPPEs. Further, the lipolysis degree of HIPPEs and the bioaccessibility of astaxanthin during in vitro digestion were improved also by the SBP microgel particles. Interestingly, the bioaccessibility of astaxanthin in HIPPEs stabilized by 4 wt% SBP microgel particles reached 51.17%. Three-dimensional (3D) printing experiments confirmed the extrudability, printing performance, and self-supporting properties of HIPPEs. In short, the HIPPEs stabilized by SBP microgel particles could be used as a delivery vehicle for astaxanthin, and the HIPPEs loaded with astaxanthin might have potential as a 3D printing material for edible functional foods.

This study aimed to evaluate the diagnostic capacity of the Nutritional Risk Screening 2002 (NRS2002), Malnutrition Universal Screening Tool (MUST), and Patient-generated Subjective Global Assessment (PG-SGA) in light of the Global Leader Initiative on Malnutrition (GLIM) criteria in adult patients with cancer. A multicenter observational study was conducted. Nutritional screening and assessment were performed at the time of admission to hospitals with the NRS2002, MUST, PG-SGA, and GLIM criteria. Sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio, and Kappa (K) values were used to evaluate the performance of these tools. Of the 637 included patients, 24.8% and 15.4% of patients were at moderate and high risk of malnutrition, respectively, using the NRS2002 and MUST. The NRS2002 was better correlated to the GLIM criteria with a higher value of Kappa (K = 0.823 vs. 0.596) and area under the receiver operating characteristic curve (K = 0.896 vs. 0.757) than the MUST. Meanwhile, 28.3% of patients were diagnosed as malnourished at the time of admission per the GLIM criteria, and 43.3% were malnourished per the PG-SGA. The PG-SGA had a fair agreement with the GLIM criteria (K = 0.453), revealing a positive predictive value of 52.9% and negative predictive value of 90.6%. The NRS2002 was better correlated with the GLIM diagnostic criteria of malnutrition than the MUST. The PG-SGA was too sensitive to detect nutrition-related deteriorations, leading to a low positive predictive value in the malnutrition diagnosis. Thus, the GLIM criteria could be used to confirm the presence of malnutrition identified by the PG-SGA in adults with cancer.

The study has been investigated on conceptualizing the state of the art of cooperate social responsibility (CSR) in green construction and its nexus to sustainable development. The research objective is to find out the significant relationship between CSR and green construction and further link green construction with sustainable development. The data has been collected from the 319 respondents working on different projects in the construction industry of China. The study is quantitative by nature. SEM analysis with the help of Smart-PLS has been applied to test the hypothesis relationship and mediations between components of CSR, green construction, and sustainable development. Results define that green procurement as a component of green construction strongly mediates between corporate social responsibility and sustainable development, and green design and CO 2 emission moderately mediates between corporate social responsibility and sustainable development. This research would add values, benefits, and knowledge toward reducing environmental temperature with the help of green construction occupying the effects of CSR on it. It would be helpful to change the trends in the construction industry to make environmental health protective and to boost the sustainable development.

Bulk nanobubbles (BNBs) are submicron gaseous domains dispersed in solutions, which are supposed to survive for several hours or even days. In recent years, there has been a rapid growth in the research and extraordinary applications of BNBs. Conventional theories based on gas diffusion and Laplace pressure, however, predicted that nanoscale gas bubbles in water should dissolve within microseconds, presenting a modern-day paradox in current nanobubbles researches. Also, it is still challenging to efficiently produce BNBs and determine their gaseous nature with the available techniques. In this review, we start from a general introduction and brief history of nanobubbles researches and revisit the current progress on the generation methods and detection techniques. Two possible formation mechanisms are suggested, and the plausibility of the proposed theories on BNBs stability is discussed with some suggestions for future studies on bulk nanobubbles.

Abstract The poor stability of Ru-based acidic oxygen evolution (OER) electrocatalysts has greatly hampered their application in polymer electrolyte membrane electrolyzers (PEMWEs). Traditional understanding of performance degradation centered on influence of bias fails in describing the stability trend, calling for deep dive into the essential origin of inactivation. Here we uncover the decisive role of reaction route (including catalytic mechanism and intermediates binding strength) on operational stability of Ru-based catalysts. Using MRuO x (M = Ce 4+ , Sn 4+ , Ru 4+ , Cr 4+ ) solid solution as structure model, we find the reaction route, thereby stability, can be customized by controlling the Ru charge. The screened SnRuO x thus exhibits orders of magnitude lifespan extension. A scalable PEMWE single cell using SnRuO x anode conveys an ever-smallest degradation rate of 53 μV h −1 during a 1300 h operation at 1 A cm −2 .

Catalyst/support interaction plays a vital role in catalysis towards acidic oxygen evolution (OER), and the performance reinforcement is currently interpreted by either strain or electron donation effect. We herein report that these views are insufficient, where the dynamic evolution of the interface under potential bias must be considered. Taking Nb2 O5-x supported iridium (Ir/Nb2 O5-x ) as a model catalyst, we uncovered the dynamic migration of oxygen species between IrOx and Nb2 O5-x during OER. Direct spectroscopic evidence combined with theoretical computation suggests these migrations not only regulate the in situ Ir structure towards boosted activity, but also suppress its over-oxidation via spontaneously delivering excessive oxygen from IrOx to Nb2 O5-x . The optimized Ir/Nb2 O5-x thus demonstrated exceptional performance in scalable water electrolyzers, i.e., only need 1.839 V to attain 3 A cm-2 (surpassing the DOE 2025 target), and no activity decay during a 2000 h test at 2 A cm-2 .

Patients with locally advanced colorectal cancer (LACRC) have a high risk of recurrence and metastasis, although neoadjuvant therapy may provide some benefit. However, patients with high microsatellite instability/deficient mismatch repair (MSI-H/dMMR) LACRC receive little benefit from neoadjuvant chemoradiotherapy (nCRT) or neoadjuvant chemotherapy (nCT). The 2015 KEYNOTE-016 trial identified MSI-H/dMMR as a biomarker indicative of immunotherapy efficacy, and pointed to the potential use of immune checkpoint inhibitors (ICIs). In 2017, the FDA approved two ICIs (pembrolizumab and nivolumab) for treatment of MSI-H/dMMR metastatic CRC (mCRC). In 2018, the CheckMate-142 trial demonstrated successful treatment of mCRC based on "double immunity" provided by nivolumab with ipilimumab, a regimen that may become a standard first-line treatment for MSI-H mCRC. In 2018, the FDA approved nivolumab alone or with ipilimumab for patients who progressed to MSI-H/dMMR mCRC after standard chemotherapy. The FDA then approved pembrolizumab alone as a first-line treatment for patients with MSI-H/dMMR CRC that was unresectable or metastatic. There is now interest in using these drugs in neoadjuvant immunotherapy (nIT) for patients with MSI-H/dMMR non-mCRC. In 2020, the NICHE trial marked the start of using nIT for CRC. This novel treatment of MSI-H/dMMR LACRC may change the approaches used for neoadjuvant therapy of other cancers. Our review of immunotherapy for CRC covers diagnosis and treatment, clinical prognostic characteristics, the mechanism of nIT, analysis of completed prospective and retrospective studies, and ongoing clinical trials, and the clinical practice of using nIT for MSI-H/dMMR LACRC. Our team also proposes a new organ-preservation strategy for patients with MSI-H/dMMR low LARC.

Despite its central importance in cellular metabolism, many details remain to be determined regarding subcellular lactate metabolism and its regulation in physiology and disease, as there is sensitive spatiotemporal resolution of lactate distribution, and dynamics remains a technical challenge. Here, we develop and characterize an ultrasensitive, highly responsive, ratiometric lactate sensor, named FiLa, enabling the monitoring of subtle lactate fluctuations in living cells and animals. Utilizing FiLa, we demonstrate that lactate is highly enriched in mammalian mitochondria and compile an atlas of subcellular lactate metabolism that reveals lactate as a key hub sensing various metabolic activities. In addition, FiLa sensors also enable direct imaging of elevated lactate levels in diabetic mice and facilitate the establishment of a simple, rapid, and sensitive lactate assay for point-of-care clinical screening. Thus, FiLa sensors provide powerful, broadly applicable tools for defining the spatiotemporal landscape of lactate metabolism in health and disease.

Orally targeted strategy of anti-inflammatory agents has attracted tremendous attention for reducing highly health-care costs and enhancing the intervention efficiency of ulcerative colitis (UC). Herein, we developed a new kind of sequence-targeted astaxanthin nanoparticles for UC treatment. Astaxanthin nanoparticles were firstly designed by self-assembly method using (3-carboxypentyl) (triphenyl) phosphonium bromide (TPP)-modified whey protein isolate (WPI)-dextran (DX) conjugates. Subsequently, lipoic acid (LA) modified hyaluronic acid (HA) was coated on the surface of the nanoparticles by double emulsion evaporation method. Exhilaratingly, the constructed sequence-targeted astaxanthin nanoparticle exhibited excellent macrophages and mitochondria targeting ability, with a Pearson's correlation coefficient of 0.84 adstnd 0.92, respectively. In vivo imaging elucidated an obvious accumulation of the sequence-targeted nanoparticles in colon tissues in UC mice. Meanwhile, the reduction stimulus release features of astaxanthin were observed in the presence of 10 mM of glutathione (GSH) at pH 7.4. Most importantly, in vivo experiments indicated that sequence-targeted astaxanthin nanoparticles could markedly alleviate inflammation by moderating the TLR4/MyD88/NF-κB signaling pathway. What's more, the composition of gut microbiota and the production of short chain fatty acid were also improved upon the uptake of sequence-targeted astaxanthin nanoparticles. Our results suggested this novel astaxanthin nanoparticles, which showed sequence-targeted ability and reduction response feature, could be exploited as a promising strategy for effective UC treatment.

The protective effect of sea bass protein (SBP)-(-)-epigallocatechin-3-gallate (EGCG) covalent complex-stabilized high internal phase (algal oil) Pickering emulsions (HIPPEs) on astaxanthin and algal oils was demonstrated in this study. The SBP-EGCG complex with better wettability and antioxidant activity was formed by the free radical-induced reaction to stabilize HIPPEs. Our results show that the SBP-EGCG complex formed dense particle shells surrounding the oil droplets, and the shells were crosslinked with the complex in the continuous phase to produce a network structure. The rheological analysis demonstrated that the SBP-EGCG complex endowed HIPPEs with high viscoelasticity, high thixotropic recovery, and good thermal stability, which were beneficial for three-dimensional (3D) printing applications. HIPPEs stabilized by SBP-EGCG complex were applied to improve the stability and bioaccessibility of astaxanthin and to delay algal oil lipid oxidation. The HIPPEs might become a food-grade 3D printing material served as a delivery system for functional foods.

Fe-Co based oxides demonstrate excellent Fenton-like catalytic activity, but the intrinsic mechanism of Fe-Co synergy remains elusive. Here, we uncover the influence of covalency competition between Fe and Co on catalytic behaviors in peroxymonosulfate (PMS) activation. Theoretical and experimental analysis reveals that higher electron delocalization of octahedral Co (CoOh)-O unit and lower electron delocalization of FeOh-O unit boost electron transfer between Co and O species and transformation of SO5•− towards surface-bounded and free SO4•− instead of 1O2. With favorable covalency competition, electron transfer capability, active oxidizing species, (Co0.8Fe0.2)Oh-O unit exhibits superior activity with rate constant of 0.27 min−1, 254.7, 37.5 and 2.2 times of that in PMS, FeOh-O unit/PMS and CoOh-O unit/PMS systems, respectively, outperforming all the reported Fe-Co based oxides in removal of refractory pollutants. The improved insights into the origin of Fe-Co synergy advance the rational development of highly efficient catalytic oxidation systems for water decontamination.

The recycling of electroplating sludge is of great significance to alleviate environmental pollution and resource depletion. However, due to the complex composition of electroplating sludge, especially chromium-containing electroplating sludge is difficult to treat, and the methods for detoxification and resource utilization of chromium-containing electroplating sludge are still lacking. Herein, we extracted chromium from electroplating sludge by a facile and economical roasting and acid leaching method to achieve detoxification, and the remaining detoxification residue was converted into a Fenton-like catalyst, which was used for the degradation of tetracycline to achieve resource utilization. The results showed that the extraction of chromium by this method could reach 93.35%, the resulting catalyst had excellent degradation performance over a wide pH range (3−11), and the removal of tetracycline could reach 98.8% under optimal conditions. Furthermore, cost calculations have shown that this method has cost advantages over traditional processing methods. In this work, an excellent performance catalyst was obtained that can be used for tetracycline degradation along with the detoxification of electroplating sludge, which provides a new route for the harmless treatment and resource utilization of electroplating sludge, with important theoretical fundamental value and potential application prospect.

ABSTRACT Cationic bactericidal peptides are components of natural host defenses against infections. While the mode of antibacterial action of cationic peptides remains controversial, several targets, including the cytoplasmic membrane and macromolecular synthesis, have been identified for peptides acting at high concentrations. The present study identified peptide effects at lower, near-lethal inhibitory concentrations. An amidated hybrid of the flounder pleurocidin and the frog dermaseptin (P-Der), two other pleurocidin derivatives, and pleurocidin itself were studied. At 2 μg/ml, the MIC, P-Der inhibited Escherichia coli growth in a broth dilution assay but did not cause bacterial death within 30 min, as estimated by viable count analysis. Consistent with this, P-Der demonstrated a weak ability to permeabilize membranes but was able to translocate across the lipid bilayer of unilamellar liposomes. Doses of 20 μg/ml or more reduced bacterial viable counts by about 2 log orders of magnitude within 5 min after peptide treatment. Abrupt loss of cell membrane potential, observed with a fluorescent dye, dipropylthiacarbocyanine, paralleled bacterial death but did not occur at the sublethal, inhibitory concentrations. Both lethal and sublethal concentrations of P-Der affected macromolecular synthesis within 5 min, as demonstrated by incorporation of [ 3 H]thymidine, [ 3 H]uridine, and [ 3 H]histidine, but the effects were qualitatively distinct at the two concentrations. Variations of the inhibition pattern described above were observed for pleurocidin and two other derivatives. Our results indicate that peptides at their lowest inhibitory concentrations may be less capable of damaging cell membranes, while they maintain their ability to inhibit macromolecular synthesis. Better understanding of the effects of peptides acting at their MICs will contribute to the design of new peptides effective at lower, less toxic concentrations.

ABSTRACT Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides. The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa , with MICs of 0.125 to 0.25 and 8 μg/ml, respectively. These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P. aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization. These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.

3-<i>O</i>-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellular production of the critical active structure is controlled by the rate-limiting enzyme, heparan sulfate d-glucosaminyl 3-<i>O</i>-sulfotransferase-1 (3-OST-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base with the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to isolate full-length cDNAs. Clones corresponding to predominant transcripts were obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3_A, and 3-OST-3_B, respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous carboxyl-terminal sulfotransferase domain of ∼260 residues. Also recovered were partial length clones for 3-OST-4. Expression of the full-length enzymes confirms the 3-<i>O</i>-sulfation of specific glucosaminyl residues within heparan sulfate (Liu, J., Shworak, N. W., Sinaÿ, P., Schwartz, J. J. Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) <i>J. Biol. Chem.</i> 274, 5185–5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 genes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy each in mice. Intriguingly, the entire sulfotransferase domain sequence of the 3-OST-3_B cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3_A. Together, these data argue that the structure of this functionally important region is actively maintained by gene conversion between 3OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified the loci for mouse<i>3Ost</i> genes and syntenic assignments of corresponding human isologs were confirmed by the identification of mapped sequence-tagged site markers. Northern blot analyses indicate brain exclusive and brain predominant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas, 3-OST-3_A and 3-OST-3_Bisoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferase domain suggest that this structure is a self-contained functional unit. Moreover, the extensive number of 3OST genes with diverse expression patterns of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST-1, regulate important biologic properties of heparan sulfate proteoglycans.

A facile dual-templating approach is demonstrated to prepare hierarchically ordered macro-/mesoporous carbons. Monodispersed silica colloidal crystals are used as a hard template, amphiphilic triblock copolymer PEO−PPO−PEO as a soft template, and soluble resols as a carbon source. The procedure involves packing and aging of silica microspheres, evaporation-induced organic−organic assembly of mesostructures in the void of microspheres, thermosetting and carbonization of phenolic formaldehyde (PF), and removal of silica scaffolds by HF. The obtained porous carbons have a highly ordered face-centered cubic macrostructure with tunable pore sizes of 230−430 nm and interconnected windows with a size of 30−65 nm. The rigid silica hard templates can prevent the shrinkage of the mesostructure during the thermosetting and carbonization process, resulting in large cell parameters (∼18 nm) and pore sizes (∼11 nm). The bimodal porous carbon materials have large BET surface areas (up to 760 m2/g), large pore volumes (∼1.25 cm3/g), and partially graphitized frameworks. With the increase in the silica sphere diameter, the BET surface areas and the window sizes increase. The hierarchically ordered carbon structures show strong diffraction at wavelengths of 500−690 nm depending on the treatment used.

Recent studies have revealed a correlation between amino acid sequences around glycosylation sites in proteoglycans and the ability of cells to initiate and process glycosaminoglycan chains. Initiation depends on Ser—Gly/Ala dipeptides that have one or more acidic amino acids in close proximity. The formation of heparan sulfate chains depends on a nearby cluster of acidic residues, hydrophobic amino acids, and the close spacing of glycosylation sites.

As a part of long-term project aimed at super polyolefin blends, in this work, we report the toughness and phase morphology of polypropylene (PP)/EPDM/SiO2 ternary composites. Two processing methods were employed to prepare PP/elastomer/filler ternary composites. One was called one-step processing method, in which the elastomer and the filler directly melt blended with PP matrix. Another one was called two-step processing method, in which the elastomer and the filler were mixed first, and then melt blended with pure PP. Two kinds of PP (grafted without or with maleic anhydride (PP-g-MA)) and SiO2 (treated with or without coupling agent) were used to control the interfacial interaction among the components. The dependence of the phase morphology on interfacial interaction and processing method was investigated. It was found that the formation of filler-network structure could be a key for a simultaneous enhancement of toughness and modulus of PP and its formation seemed to be dependent on the work of adhesion (WAB) and processing method. As the WAB of PP/EPDM interface was much lower than that of PP/SiO2 and EPDM/SiO2, and the two-step processing method was used, the formation of filler-network structure was favorable. In this case, a super toughened PP ternary composite with the Izod impact strength 2–3 times higher than PP/EPDM binary blend and 15–20 times higher than pure PP could be achieved.

Purpose To study the histopathologic features of CT screen-detected Stage IA adenocarcinomas to determine whether survival differed by the proportion of bronchioloalveolar component (BAC) or by the presence of multiple lesions in node-negative patients. Methods Five pathologists with expertise in pulmonary pathology examined 279 resected cases of adenocarcinomas, 30 mm or less in length diagnosed by CT screening for lung cancer. The panel determined the consensus diagnosis for each case, identified additional cancers, and classified each case as solitary or non-solitary. The presence and proportion of BAC was also documented. Results Of the cases of adenocarcinoma, 20 (7%) were BAC subtype, 246 (88%) mixed subtype and 13 (5%) adenocarcinoma-OTHER. BAC cases manifested as non-solid and part solid nodules, mixed as solid and part-solid, and other as solid only. Kaplan–Meier 10-year survival rates were 100% for BAC and adeno-MIXED with 90–99% BAC cases, 95% for mixed with 1–90% BAC, 90% for those without a BAC component, and 75% for other cases. Fifty (18%) cases were non-solitary carcinomas and 44 of these were node negative; the non-solitary node-negative cases had the same excellent prognosis as solitary node-negative cases. Conclusions The proportion of BAC component was a positive prognostic factor and correlated with CT consistency. Contrary to staging predictions, cases of non-solitary node-negative adenocarcinoma had the same excellent prognosis as solitary node-negative cases, suggesting that most of the small, node-negative multiple carcinomas probably represent multiple primaries rather than intrapulmonary metastasis.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-β (Aβ) peptides in the brain are central to its pathogenesis. In healthy brains, Aβ is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of Aβ is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of Aβ in neurons, glia cells, and along the brain vasculatures. Heparan sulfate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its colocalization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular Aβ uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses Aβ uptake, whereas overexpression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular Aβ uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of Aβ. We further showed that HSPG is more important for the binding of Aβ to the cell surface than LRP1. The critical roles of HSPG in cellular Aβ binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed Aβ uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular Aβ uptake and define a major pathway through which Aβ gains entry to neuronal cells.

Highly ordered mesoporous carbon−titania nanocomposites with nanocrystal-glass frameworks have been synthesized via the organic−inorganic−amphiphilic coassembly followed by the in situ crystallization technology. A soluble resol polymer was used as a carbon precursor, prehydrolyzed TiCl4 as an inorganic precursor, and triblock copolymer F127 as a template. The carbon−titania nanocomposites with controllable texture properties and composition can be obtained in a wide range from 20 to 80 wt% TiO2 by adjusting the initial mass ratios. The C−TiO2 nanocomposites with "bricked-mortar"frameworks exhibit highly ordered 2D hexagonal mesostructure and high thermal stability up to 700 °C. The nanocomposites have high surface area (465 m2 g−1) and large pore size (∼4.1 nm). Additionally, the nanocomposites show good performance in degradation of Rhodamine B due to the photocatalytic activity of the titania nanocrystals and the strong adsorptive capacity of the glasslike carbon.

Conducting nanotubes of polyaniline (PANI) about 80–180 nm in diameter were synthesized by a chemical template-free method in the presence of D-10-camphorsulfonic acid (D-CSA) as the dopant, and ammonium persulfate ((NH4)2S2O8) as the oxidant. The effect of synthetic conditions, such as the molar ratio of D-CSA to aniline (An), the concentration of D-CSA in the polymerization media, the reaction temperature and time, on the morphology and size as well as the electrical properties of the PANI–(D-CSA) was investigated. It was found that the above synthetic conditions, especially the molar ratio of D-CSA to An, strongly affected the morphology and formation probability of the resulting PANI. The micelles formed by D-CSA and anilinium cations act as the templates in the formation of PANI–(D-CSA) nanotubes.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999)J. Biol. Chem. 274, 5170–5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate d-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3′-phosphophate 5′-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide. The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate d-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999)J. Biol. Chem. 274, 5170–5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate d-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3′-phosphophate 5′-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide. The cell surface and extracellular matrix contain heparan sulfate proteoglycans which consist of a core protein and single or multiple heparan sulfate (HS) 1The abbreviations HSheparan sulfatePAPSadenosine 3′-phosphosphate 5′-phosphosulfateGlcAd-glucuronic acidGlcNAcN-acetyl-d-glucosamine3-OSTheparan sulfated-glucosaminyl 3-O-sulfotransferaseGlcNSN-sulfo-d-glucosamineIdoAl-iduronic acidHSactanticoagulant heparan sulfateHSinactnonanticoagulant heparan sulfateIdoA2Sl-iduronic acid 2-O-sulfateGlcA2Sd-glucuronic acid 2-O-sulfateGlcNR'6SN-acetyl-d-glucosamine 6-O-sulfate orN-sulfo-d-glucosamine 6-O-sulfateGlcNS3S±6SN-sulfo-d-glucosamine 3-O-sulfate or N-sulfo-d-glucosamine 3,6-O-bisulfateHPLChigh performance liquid chromatographySAX-HPLCstrong anion exchange-HPLCRPIP-HPLCreversed phase ion pairing HPLCAnMan2,5-anhydro-d-mannitolAnMan3SAnMan6S, and AnMan3S6S, 2,5-anhydro-d-mannitol 3-O-sulfate, 6-O-sulfate and 3,6-O-bisulfate, respectivelyATantithrombinMes4-morpholineethanesulfonic acid 1The abbreviations HSheparan sulfatePAPSadenosine 3′-phosphosphate 5′-phosphosulfateGlcAd-glucuronic acidGlcNAcN-acetyl-d-glucosamine3-OSTheparan sulfated-glucosaminyl 3-O-sulfotransferaseGlcNSN-sulfo-d-glucosamineIdoAl-iduronic acidHSactanticoagulant heparan sulfateHSinactnonanticoagulant heparan sulfateIdoA2Sl-iduronic acid 2-O-sulfateGlcA2Sd-glucuronic acid 2-O-sulfateGlcNR'6SN-acetyl-d-glucosamine 6-O-sulfate orN-sulfo-d-glucosamine 6-O-sulfateGlcNS3S±6SN-sulfo-d-glucosamine 3-O-sulfate or N-sulfo-d-glucosamine 3,6-O-bisulfateHPLChigh performance liquid chromatographySAX-HPLCstrong anion exchange-HPLCRPIP-HPLCreversed phase ion pairing HPLCAnMan2,5-anhydro-d-mannitolAnMan3SAnMan6S, and AnMan3S6S, 2,5-anhydro-d-mannitol 3-O-sulfate, 6-O-sulfate and 3,6-O-bisulfate, respectivelyATantithrombinMes4-morpholineethanesulfonic acidside chains. The highly charged HS side chains are the copolymers of glucuronic acid (GlcA)/iduronic acid (IdoA) andN-acetylated glucosamine (GlcNAc) with various sulfations. HS binds to specific proteins such as antithrombin and several growth factors, thereby regulates various biological processes including anticoagulation and angiogenesis (1Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (254) Google Scholar). The unique sulfation pattern of a monosaccharide sequence within HS is believed to be critical for binding to the target protein. The biosynthesis of HS involves the formation of a polysaccharide backbone followed by serial sulfation and epimerization reactions. The HS backbone is elongated by HS copolymerase-dependent (2Lind T. Lindahl U. Lidholt K. J. Biol. Chem. 1993; 268: 20705-20708Abstract Full Text PDF PubMed Google Scholar) transfer of GlcA and GlcNAc to the tetrasaccharide linkage region present on core proteins. This process creates a polysaccharide backbone of approximately 100 repeating disaccharide units (1Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (254) Google Scholar). This polysaccharide is structurally altered at GlcNAc to form N-sulfated glucosamine (GlcNS) by heparan sulfate N-deacetylase/N-sulfotransferase (3Orellana A. Hirschberg C.B. Wei Z. Swiedler S.J. Ishihara M. J. Biol. Chem. 1994; 269: 2270-2276Abstract Full Text PDF PubMed Google Scholar, 4Hashimoto Y. Orellana A. Gil G. Hirschberg C.B. J. Biol. Chem. 1992; 267: 15744-15750Abstract Full Text PDF PubMed Google Scholar, 5Eriksson I. Sandbäck D. Ek B. Lindahl U. Kjellén L. J. Biol. Chem. 1994; 269: 10438-10443Abstract Full Text PDF PubMed Google Scholar). The heparan sulfate C5 epimerase subsequently converts occasional GlcA to IdoA within the polysaccharide (6Li J.-p. Hagner-McWhirter A. Kjellén L. Palgi J. Jalkanen M. Lindahl U. J. Biol. Chem. 1997; 272: 28158-28163Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Heparan sulfate (uronosyl) 2-O-sulfotransferase transfers sulfate from PAPS to IdoA to form 2-O-sulfated iduronic acid (IdoA2S) (7Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and heparan sulfate (d-glucosaminyl) 6-O-sulfotransferase transfers sulfate to the 6-OH position of glucosamine to form 6-O-sulfated glucosamine (8Habuchi H. Habuchi O. Kimata K. J. Biol. Chem. 1995; 270: 4172-4179Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Although this mechanism provides a scheme for generating the average structure of HS, it is unable to explain how HS with defined monosaccharide sequences are produced.We have delineated a mechanism for generating HS with a specific monosaccharide sequence based on our investigation of the biosynthesis of anticoagulant active HS (HSact). Our results show that the synthesis of HSact requires a limiting factor, as well as a precursor with a defined polysaccharide structure which can be modified by the limiting factor. HSact, normally present as 1–10% of total HS, contains a structurally defined antithrombin-binding pentasaccharide with a sequence of -GlcNSorAc6S-GlcA-GlcNS3S±6S-IdoA2S-GlcNS6S- (9Lindahl U. Bäckström G. Thumberg L. Leder I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6551-6555Crossref PubMed Scopus (413) Google Scholar, 10Rosenberg R.D. Lam L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1218-1222Crossref PubMed Scopus (231) Google Scholar). Our previous work demonstrated the presence of a specific pathway for the biosynthesis of HSact in L cells. Overexpression of syndecan-4 core protein or chemical mutagenesis in wild-type L cells suggest that HSact generation requires a limiting factor, and the limiting factor was identified as heparan sulfate 3-O-sulfotransferase (3-OST-1, EC 2.8.2.23) (11Shworak N.W. Shirakawa M. Colliec-Joualt S. Liu J. Mulligan R.C. Birinyi L.K. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24941-24952Abstract Full Text PDF PubMed Google Scholar, 12Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar, 13Shworak N.W. Fritze L.M.S. Liu J. Butler L.D. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27063-27071Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Loggeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).We recently proposed that HS biosynthetic enzymes are present in multiple isoforms, and each isoform has slightly different substrate specificities and recognizes the monosaccharide sequences around the modification sites (1Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (254) Google Scholar). Indeed, the available data support this hypothesis (16Ishihara M. Guo Y. Wei Z. Yang Z. Swiedler S.J. Orellana A. Hirschberg C.B. J. Biol. Chem. 1993; 268: 20091-20095Abstract Full Text PDF PubMed Google Scholar, 17Cheung W.-F. Eriksson I. Kusche-Gullberg M. Lindahl U. Kjellén L. Biochemistry. 1996; 35: 5250-5256Crossref PubMed Scopus (49) Google Scholar, 18Fritz T.A. Gabb M.M. Wei G. Esko J.D. J. Biol. Chem. 1994; 269: 28809-28814Abstract Full Text PDF PubMed Google Scholar). As documented by several researchers including ourselves, 3-O-sulfation of glucosamine is a rare modification that constitutes the last step in the biosynthesis of HS (14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Loggeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 19Linhardt R.J. Wang H. Loganathan D. Bae J. J. Biol. Chem. 1992; 267: 2380-2387Abstract Full Text PDF PubMed Google Scholar, 20Lindahl U. Feingold D.S. Roden L. Trends Biochem. Sci. 1986; 11: 221-225Abstract Full Text PDF Scopus (104) Google Scholar). We have shown that 3-OST-1 requires an appropriate HS precursor with defined monosaccharide sequence for sulfation to occur and make HSact. 2Zhang, L., Yoshida, K., Liu, J., and Rosenberg, R. D. (1999) J. Biol. Chem. 274,in press. 2Zhang, L., Yoshida, K., Liu, J., and Rosenberg, R. D. (1999) J. Biol. Chem. 274,in press.It is likely that 3-O-sulfated HS are specifically involved in other HS-related biological processes. If this is the case, studies of 3-O-sulfation should facilitate the identification of new biologically active HS-binding proteins. The 3-OST-1, which we have previously purified and molecularly cloned, only sulfates the glucosamine in GlcA-GlcNS±6S. However, there are at least two other types of 3-O-sulfated glucosamine that were previously characterized. For example, the HS isolated from glomerular basement membrane contains substantial amounts of an IdoA2S-GlcNS3S disaccharide unit (21Edge A.S.B. Spiro R.G. J. Biol. Chem. 1990; 265: 15874-15881Abstract Full Text PDF PubMed Google Scholar, 22Shen G.-Q. Kresbach G. Spiro M.J. Spiro R.G. Arch. Biochem. Biophys. 1995; 321: 83-93Crossref PubMed Scopus (8) Google Scholar). Pejler et al. (23Pejler G. Danielsson Å. Björk I. Lindahl U. Nader H.B. Dietrich C.P. J. Biol. Chem. 1987; 262: 11413-11421Abstract Full Text PDF PubMed Google Scholar) also reported that a heparin isolated from the clams Anomalocardia brasiliana andTivela mactroides contained IdoA-GlcNS3S6S disaccharide (23Pejler G. Danielsson Å. Björk I. Lindahl U. Nader H.B. Dietrich C.P. J. Biol. Chem. 1987; 262: 11413-11421Abstract Full Text PDF PubMed Google Scholar). It is apparent that neither the IdoA2S-GlcNS3S nor the IdoA-GlcNS3S6S is the product of 3-OST-1. These observations suggest the possible existence of different heparan sulfate 3-O-sulfotransferases, which specifically sulfate the 3-OH positions of glucosamine residues that are linked to various sulfated/nonsulfated uronic acid residues at the nonreducing termini. In this article, we report the expression and characterization of new heparan sulfate 3-O-sulfotransferase isoforms, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B that were isolated as described in the accompanying article (24Shworak N.W. Liu J. Petros L.M. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). We have found that 3-OST-2 and 3-OST-3A catalyze the 3-O-sulfation of glucosamine in GlcA2S-GlcNS and IdoA2S-GlcNS, respectively, and 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. The HS modified by 3-OST-2 and 3-OST-3A do not contain anticoagulant activity. Our studies reveal the presence of different isoforms, each of which sulfates the sugar residue in unique disaccharides or possibly in oligosaccharide sequences. Thus, we provide a new mechanism for the biosynthesis of HS with specific monosaccharide sequences and different potential biological functions.EXPERIMENTAL PROCEDURESMaterialsPlasmids pJL2.7, pJL3.3, and pJL3.6 containing 3-OST-2, 3-OST-3A and 3-OST-3B were isolated from human brain and liver cDNA libraries (24Shworak N.W. Liu J. Petros L.M. Kobayashi M. Copeland N.G. Jenkins N.A. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5170-5184Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The [35S]PAPS (≈150 Ci/mmol) was prepared by incubating 0.4 mCi/ml [35S]Na2SO4 (ICN) and 16 mm ATP with 5 mg/ml dialyzed yeast extract (Sigma) as described previously (14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Iduronate 2-sulfatase, β-glucuronidase, and α-iduronidase were purified from bovine liver (10Rosenberg R.D. Lam L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1218-1222Crossref PubMed Scopus (231) Google Scholar). The following [3H]disaccharide standards were prepared from antithrombin-binding heparin octasaccharide (a gift from Dr. Lun H. Lam, Glycomed) (12Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar): GlcA-[ 3All 3H-labeled disaccharide and monosaccharide standards used in the experiments were labeled at the C1 position of 2,5-anhydromannitol. The structures of di- and oligosaccharides were presented in an abbreviated format omittingd-, l- α1→4, and β1→4 for each sugar residue in order to conserve space and improve the clarity. -GlcA-, -GlcNR‘-, and -IdoA- represents the linkage of →4)-β-d-GlcA(1→, →4)-α-d-GlcNR‘(1→, and →4)-α-l-IdoA-(1→, respectively. H]AnMan6S,3IdoA-[3H]AnMan6S, IdoA2S-[3H]AnMan, GlcA-[3H]AnMan3S, IdoA2S-[3H]AnMan6S, and GlcA-[3H]AnMan3S6S.Expression of 3-OST IsoformsThe expression plasmids for 3-OST-2 and 3-OST-3Awere constructed by inserting EcoRI/XbaI cleaved fragments from pJL 2.7 and pJL 3.3 into pcDNA3 (Invitrogen) which was digested with EcoRI/XbaI followed by phosphatase treatment. The expression plasmid for 3-OST-3Bwas similarly constructed by inserting theHindIII/NotI cleaved fragment from pJL 3.6 into pcDNA3 which was digested with HindIII/NotI. The 3-OST-1 expression construct has been previously described (15Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Loggeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The expression plasmids were transfected into exponentially growing COS-7 cells by using DEAE dextran and dimethyl sulfoxide (15Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Loggeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).Measurement of 3-OST-2 and 3-OST-3A/-3BActivitiesThe crude enzymes were extracted from transiently transfected COS-7 cell pellets by vortexing 2.5 × 105 cells in 200 μl of ice-cold 0.25 m sucrose with 1% Triton X-100 (v/v), and the insoluble residue was removed by spinning at 10,000 × g for 10 min. To determine HS sulfotransferase activity, the reaction contained 10–40 μg of cell extract protein, 200 nm unlabeled HSinact isolated from 33 cells (14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), 60 × 106 cpm of [35S]PAPS, 50 mm MES (Sigma), pH 7.0, 10 mmMnCl2, 5 mm MgCl2, 75 μg/ml protamine chloride (Sigma), 120 μg/ml bovine serum albumin, and 1% Triton X-100 (v/v) in a final volume of 50 μl. The reaction mixture was incubated at 37 °C for 2 h, quenched by heating to 100 °C for 2 min and spun at 14,000 × g for 2 min to remove insoluble material. The sample was then subjected to a 200-μl DEAE-Sepharose column to isolate [35S]HS (14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,25Shworak N.W. Shirakawa M. Mulligan R.C. Rosenberg R.D. J. Biol. Chem. 1994; 269: 21204-21214Abstract Full Text PDF PubMed Google Scholar). One unit of HS sulfotransferase activity was defined as the transfer of 1 fmol (×10−15 mol) of sulfate from PAPS to heparan sulfate under standard assay conditions. The procedure for determining HSact conversion activity was based upon our previous report (14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). One unit of the HSact conversion activity was defined as 0.5% increase of HSact per 20 min under standard conditions.Degradation of 35S-Labeled HSLow pH Nitrous Acid Degradation of [35S]HSPurified [35S]HS modified by 3-OST-2, 3-OST-3A, and 3-OST-3B (2 × 104 to 4 × 106 cpm) was mixed with 20 μg of porcine kidney HS (ICN), treated on ice with HNO2, at pH 1.5 for 30 min, and then reduced under alkaline conditions with 0.5 m NaBH4 (26Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (664) Google Scholar). The resultant35S-disaccharides were mixed with IdoA2S-[3H]AnMan6S (2 × 104 to 2 × 106 cpm) and desalted on a Bio-Gel P-2 gel column (0.75 × 200 cm) which was equilibrated with 0.1 mammonium bicarbonate at a flow rate of 4 ml/h.High Performance Liquid ChromatographySeparation and characterization of 35S-labeled disaccharides were carried out by HPLC using either a C18-reversed phase column (0.46 × 25 cm) (RPIP-HPLC) (Vydac) or a Partisil-5 strong anion exchange column (0.46 × 25 cm) (SAX-HPLC) (Whatman). The RPIP-HPLC was eluted with 4.2% acetonitrile for 45 min followed by 9% acetonitrile for 15 min and then by 10.2% acetonitrile for 120 min in 10 mm ammonium dihydrophosphate and 1 mmtetrabutylammonium dihydrophosphate (Sigma) at a flow rate of 0.5 ml/min (11Shworak N.W. Shirakawa M. Colliec-Joualt S. Liu J. Mulligan R.C. Birinyi L.K. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24941-24952Abstract Full Text PDF PubMed Google Scholar, 12Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar, 27Guo Y. Conrad H.E. Anal. Biochem. 1988; 168: 54-62Crossref PubMed Scopus (50) Google Scholar); the SAX-HPLC was eluted with 30 mmKH2PO4 for 80 min followed by a linear gradient from 30 to 400 mm KH2PO4 over 120 min at a flow rate of 0.8 ml/min (11,12).Determination of the Structures of Peak-X and Peak-YPreparation of 3H-Labeled Di- and Monosaccharide StandardsDisaccharide IdoA3S-GlcNS3SOMe (methyl-(3-O-sodium sulfonato-sodium α-l-idopyranosyluronate)-(1→4)-2-deoxy-2-sodium sulfonatamido-3-O-sodium-sulfonato-α-d-glucopyranoside) and GlcA3S-GlcNS3SOMe (methyl-(3-O-sodium sulfonato-sodium β-d-glucopyranosyluronate)-(1→4)-2-deoxy-2-sodium sulfonatamido-3-O-sodium sulfonato-α-d-glucopyranoside) were chemically synthesized (28Petitou M. Duchaussoy P. Lederman I. Choay J. Jacquinet J.-C. Sinaÿ P. Torri G. Carbohydr. Res. 1987; 167: 67-75Crossref PubMed Scopus (140) Google Scholar). The structures of these two compounds were confirmed by 1H NMR (2H2O, 250 Mhz). 4S. Tripathy, J.-M. Mallet, M. Petitou, and P. Sinaÿ, manuscript in preparation. IdoA3S-[3H]AnMan3S and GlcA3S-[3H]AnMan3S were prepared by low pH nitrous acid treatment of the corresponding chemically synthesized disaccharide followed by a reduction with sodium [3H]borohydride. GlcA2S-[3H]AnMan6S was prepared by epimerization of IdoA2S-[3H]AnMan6S in the presence of hydrazine and hydrazine sulfate. The products were confirmed by chemical desulfation in the presence of 0.5 msulfuric acid followed by β-glucuronidase digestion. [3H]AnMan3S was prepared from β-d-glucosamine 3-O-sulfate (Sigma) by nitrous acid treatment at pH 1.5 followed by NaB3H4reduction. [3H]AnMan6S was prepared from β-glucuronidase-treated GlcA-[3H]AnMan6S.Epimerization and Chemical Desulfation of 35S- and3H-Labeled Disaccharides35S-Labeled disaccharides (5–10 × 104 cpm) were mixed with appropriate 3H-labeled disaccharide standards (1–5 × 105 cpm) in 50 μl of anhydrous hydrazine (Aldrich) containing 10 mg/ml hydrazine sulfate and incubated at 100 °C in a sealed 1.0-ml vacuum hydrolysis tube (Pierce) for 16 h followed by HIO3 oxidation as described by Shaklee and Conrad (29Shaklee P.N. Conrad H.E. Biochem. J. 1984; 217: 187-197Crossref PubMed Scopus (77) Google Scholar). The partial acid hydrolysis of 35S-labeled disaccharide (2 × 104 cpm) was performed in 0.5 mH2SO4 and incubated at 100 °C for 15 min as described elsewhere (30Delaney S.R. Leger M. Conrad H.E. Anal. Biochem. 1980; 100: 253-261Crossref Scopus (30) Google Scholar). The partially desulfated [35S]disaccharide became susceptible to α-iduronidase or β-glucuronidase which permitted the establishment of the structure of the disaccharide as well as the position of [35S]sulfate (14,31).β-Glucuronidase and α-Iduronidase Digestion35S-Labeled disaccharide (1 × 104 cpm) was mixed with GlcA-[3H]AnMan6S or GlcA-[3H]AnMan3S6S in 50 μl of 50 mm NaAcO, pH 4.5, and 100 μl of purified β-glucuronidase (5,000 units) and incubated at 37 °C for 36 h. For α-iduronidase digestion, the35S-labeled disaccharide was mixed with IdoA-[3H]AnMan6S (1–10 × 104 cpm) in 50 μl of buffer containing 250 mm sodium formate and 400 mm NaCl, pH 3.55, and 100 μl of α-iduronidase (8 units) and incubated at 37 °C for 36 h.Iduronate 2-Sulfatase DigestionBecause iduronate 2-sulfatase was extremely sensitive to phosphate (IC50 = 25–100 μm) (32Bielicki J. Freeman C. Clements P.R. Hopwood J.J. Biochem. J. 1990; 271: 75-86Crossref PubMed Scopus (71) Google Scholar), the disaccharide samples were desalted extensively by using a combination of DEAE chromatography, P-2 gel, and descending paper chromatography. Briefly, the RPIP-HPLC purified [35S]disaccharides were diluted 5–10-fold with water to bring the concentration of phosphate to less than 5 mm, neutralized with 10 m NaOH and charged to a 1-ml DEAE-Sephacel column (Sigma), which was equilibrated with 10 mm triethylammonium bicarbonate buffer (Sigma), pH 8.0. The column was washed with 10 × 1 ml of 100 mmtriethylammonium bicarbonate, pH 8.0, and eluted with 400 mm triethylammonium bicarbonate, pH 8.0. The resultant disaccharides were loaded on a P-2 gel column (0.75 × 200 cm) equilibrated and eluted with 0.1 m ammonium bicarbonate at a flow rate of 4 ml/h. The disaccharides were then loaded on 3MM paper (Whatman) which was developed in ethyl acetate/formic acid/acetic acid/water = 36:2:6:1 for 2 h (12Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar, 13Shworak N.W. Fritze L.M.S. Liu J. Butler L.D. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27063-27071Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and the disaccharides were recovered by eluting from the 3MM paper with 3 × 0.2 ml of water. The condition for iduronate 2-sulfatase digestion followed the procedures reported by Bielicki et al. (32Bielicki J. Freeman C. Clements P.R. Hopwood J.J. Biochem. J. 1990; 271: 75-86Crossref PubMed Scopus (71) Google Scholar).DISCUSSIONThe cDNAs, which have approximately 60% similarity to the earlier cloned 3-OST-1 in the sulfotransferase domain, were demonstrated to encode heparan sulfate 3-O-sulfotransferase isoforms. The strategy used to characterize these cDNAs is the following: 1) expression of the cDNAs in COS-7 cells by transient transfection; 2) preparation of 35S-labeled HS by mixing the [35S]PAPS and unlabeled HS with the COS-7 cell extracts transfected with 3-OST-2, 3-OST-3A, and 3-OST-3B; 3) determination of the 35S labeling site within the [35S]HS by degrading the HS with nitrous acid at low pH (pH 1.5). A similar approach has been previously used to characterize 3-OST-1, heparan sulfate (d-glucosaminyl) 6-O-sulfotransferase, and heparan sulfate (uronosyl) 2-O-sulfotransferase (7Kobayashi M. Habuchi H. Yoneda M. Habuchi O. Kimata K. J. Biol. Chem. 1997; 272: 13980-13985Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 8Habuchi H. Habuchi O. Kimata K. J. Biol. Chem. 1995; 270: 4172-4179Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Our data suggest that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate 3-O-sulfotransferases because they transfer [35S]sulfate from [35S]PAPS to the 3-OH position of the glucosamine residue, and 3-OST-3A and 3-OST-3B sulfates an identical disaccharide.To our knowledge, the activities of 3-OST-2 and 3-OST-3Ahave not been previously reported. The substrate specificities of 3-OST-2 and 3-OST-3A at the disaccharide level were identified by determining the structures and [35S]sulfate labeling sites of two 35S-labeled disaccharides designated as Peak-X and Peak-Y. These two disaccharides are the products of low pH nitrous acid-treated [35S]HS prepared by 3-OST-2 and 3-OST-3A. Peak-X was identified to be IdoA2S-[35S]AnMan3S, and Peak-Y was identified to be GlcA2S-[35S]AnMan3S. The structure GlcA2S-AnMan3S has not been identified previously in naturally occurring heparin and heparan sulfate. However, because of the potential side reactions during chemical degradation of [35S]HS, the substrate specificities of 3-OST-2 and 3-OST-3A have not been conclusively determined. Such side reactions can only be controlled by using chemically defined oligosaccharides with expected modifications.The differences between the isoforms and 3-OST-1 are as follows: 3-OST-1 generates HSact, while 3-OST-2 and 3-OST-3A do not. In addition, the isoforms transfer sulfate to uronyl-glucosamine disaccharides with different sulfated uronic acid residues. For example, 3-OST-1 transfers sulfate to the 3-OH position of the glucosamine within GlcA-GlcNS ± 6S; 3-OST-2 transfers sulfate to the 3-OH position of the glucosamine within GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to the 3-OH position of the glucosamine within IdoA2S-GlcNS. However, we cannot rule out the possibility that 3-OST-2 and 3-OST-3A sulfate the 3-OH position of the glucosamine within GlcA2S-GlcNH2and IdoA2S-GlcNH2, respectively. 6We have recently found that purified 3-OST-3A sulfates the glucosamine residue with free amino group. The conclusion was based on the results of the structural analysis of two 35S-labeled tetrasaccharides, which were isolated from heparin lyases degraded the [35S]HS [35S]sulfated by purified 3-OST-3A. Furthermore, 3-OST-1 is a secretary protein, because substantial amounts of the activity were detected in the cell medium and no p

Core-shell polystyrene-silica spheres with diameters of 800 nm and 1.9 microm were synthesized by soap-free emulsion and dispersion polymerization of the polystyrene core, respectively. The polystyrene spheres were used as templates for the synthesis of silica shells of tunable thickness employing the Stöber method [Graf et al. Langmuir 2003, 19, 6693]. The polystyrene template was removed by thermal decomposition at 500 degrees C, resulting in smooth silica shells of well-defined thickness (15-70 nm). The elastic response of these hollow spheres was probed by atomic force microscopy (AFM). A point load was applied to the particle surface through a sharp AFM tip, and successively increased until the shell broke. In agreement with the predictions of shell theory, for small deformations the deformation increased linearly with applied force. The Young's modulus (18 +/- 6 GPa) was about 4 times smaller than that of fused silica [Adachi and Sakka J. Mater. Sci. 1990, 25, 4732] but identical to that of bulk silica spheres (800 nm) synthesized by the Stöber method, indicating that it yields silica of lower density. The minimum force needed to irreversibly deform (buckle) the shell increased quadratically with shell thickness.

Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease in China. A cluster of cases among health care workers and family members following exposure to a patient with fulminant disease consistent with HGA prompted investigation.To investigate the origin and transmission of apparent nosocomial cases of febrile illness in the Anhui Province.After exposure to an index patient whose fatal illness was characterized by fever and hemorrhage at a primary care hospital and regional tertiary care hospital's isolation ward, secondary cases with febrile illness who were suspected of being exposed were tested for antibodies against Anaplasma phagocytophilum and by polymerase chain reaction (PCR) and DNA sequencing for A. phagocytophilum DNA. Potential sources of exposure were investigated.Cases with serological or PCR evidence of HGA were compared with uninfected contacts to define the attack rate, relative risk of illness, and potential risks for exposure during the provision of care to the index patient.In a regional hospital of Anhui Province, China, between November 9 and 17, 2006, a cluster of 9 febrile patients with leukopenia, thrombocytopenia, and elevated serum aminotransferase levels were diagnosed with HGA by PCR for A. phagocytophilum DNA in peripheral blood and by seroconversion to A. phagocytophilum. No patients had tick bites. All 9 patients had contact with the index patient within 12 hours of her death from suspected fatal HGA while she experienced extensive hemorrhage and underwent endotracheal intubation. The attack rate was 32.1% vs 0% (P = .04) among contacts exposed at 50 cm or closer, 45% vs 0% (P = .001) among those exposed for more than 2 hours, 75% vs 0% (P < .001) among those reporting contact with blood secretions, and 87.5% vs 0% (P = .004) among those reporting contact with respiratory secretions from the index patient.We report the identification of HGA in China and likely nosocomial transmission of HGA from direct contact with blood or respiratory secretions.

China is the world's largest emitter of greenhouse gases (GHGs) and the agricultural sector in China is responsible for 17–20% of annual emissions and 62% of total freshwater use. Groundwater abstraction in China has increased rapidly from 10 km3 yr−1 in the 1950s to more than 100 km3 yr−1 in the 2000s, such that roughly 70% of the irrigated area in northern China is now groundwater-fed. Pumping of water for irrigation is one of the most energy consuming on-farm processes; however, to date this source of GHG emissions in China and elsewhere has been relatively neglected. We derive the first detailed estimate of GHG emissions from groundwater pumping for irrigation in China, using extensive village survey data from 11 provinces, broadly representative of the situation during the mid-2000s. The 11 provinces cover roughly half of China's irrigated cropland and we upscale to the national level using government statistics for the remaining 20 provinces. Our results show emissions of 33.1 MtCO2e, just over half a per cent of the national total. Groundwater abstraction represents an important source of GHG emissions that has been rapidly increasing and which at present is largely unregulated. Water scarcity in China is already driving policies to improve water conservation. These results suggest that significant potential exists to promote the co-benefits of water and energy saving in order to meet national planning targets.

Heparan sulfate, a cell surface bound glycosaminoglycan polysaccharide, has been implicated in numerous biological functions. Heparan sulfate molecules are highly complex and diverse, yet deceivingly look simple and similar, rendering structure--function correlation tedious. Current chromatographic and mass spectrometric techniques have limitations for analyzing glycosaminoglycan samples that are in low abundance and that are large in size, due to their highly acidic nature arising from a large number of sulfate and of carboxylate groups. A new methodology was developed using capillary ion-paired reverse-phase C18 HPLC directly coupled to ESI-TOF-MS to address the above issues. On the basis of HS disaccharide analysis, dibutylamine was found to be the best suited for HS analysis among many ion-pairing agents investigated. Next, analysis of oligosaccharides derived from heparosan, the precursor for heparan sulfate, was undertaken to demonstrate its greater applicability in a more complex structural analysis. The established chromatographic conditions enabled the characterization of heparosan oligosaccharides of sizes up to tetracontasaccharide with high resolution in a single run and were amenable to negative ion electrospray MS in which sodium adduction and fragmentation were avoided. To date, these are the largest nonsulfated HS precursor oligosaccharides to be characterized by LC/MS. Finally, the current methodology was applied to the characterization of the biologically important ATIII binding pentasaccharide and its precursors, which differ from each other by sulfation pattern and/or degree of sulfation. All of these pentasaccharides were well-resolved and characterized by the LC/MS system with (34)SO(4) as a mass spectral probe. This newly developed methodology facilitates the purification and rapid characterization of biologically significant HS oligosaccharides, and will thus expedite their synthesis. These findings should undoubtedly pave the way in deciphering multiple functional arrangements, ascribed to many biological activities, which are predictably embedded in a single large chaotic, yet well-organized HS polysaccharide chain. Development of newer techniques for HS oligosaccharide analysis is greatly needed in the postgenome era as attention shifts to the functional implications of proteins and carbohydrates in general and HS in particular.

Greater than 90% of lung infections in cystic fibrosis (CF) patients are caused by Pseudomonas aeruginosa, and the majority of these patients subsequently die from lung damage. Current therapies are either targeted at reducing obstruction, reducing inflammation, or reducing infection. To identify potential therapeutic agents for the CF lung, 150 antimicrobial peptides consisting of three distinct structural classes were screened against mucoid and multidrug-resistant clinical isolates of P. aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Staphylococcus aureus. Five peptides that retained potent antimicrobial activities in physiological salt and divalent cation environment were further characterized in vivo using a rat chronic lung infection model. All animals were inoculated intratracheally with 10(4) P. aeruginosa mucoid PAO1 cells in agar beads. Three days following inoculation treatment was initiated. Animals were treated daily for 3 days with 100 microl of peptide solution (1 mg/ml) in 10 mM sodium citrate, which was deposited via either intratracheal instillation or aerosolization. Control animals received daily exposure to vehicle alone. At the end of the treatment the lungs of the animals were removed for quantitative culture. Four peptides, HBCM2, HBCM3, HBCPalpha-2, and HB71, demonstrated significant reduction in Pseudomonas bioburden in the lung of rats. Further in vivo studies provided direct evidence that anti-inflammatory activity was associated with three of these peptides. Therefore, small bioactive peptides have the potential to attack two of the components responsible for the progression of lung damage in the CF disease: infection and inflammation.

A novel strategy for the fabrication of sensitive immunosensor to detect α-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml−1 with a detection limit of 0.6 ng ml−1. The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP–anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.

ABSTRACT While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Using influenza A virus matrix protein 1 (M1 58-66 ) epitope-specific CTLs isolated from healthy HLA-A2 + individuals, we further found that M1 58-66 epitope-specific CTLs efficiently killed both M1 58-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M1 58-66 -specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M1 58-66 epitope-specific CTLs in 20% (4/20) of HLA-A2 + individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.

With the increasing applications of metal-based nanoparticles in various commercial products, it is necessary to address their environmental fate and potential toxicity. In this work, we assessed the phytotoxicity of lanthanum oxide (La2O3) NPs to cucumber plants and determined its distribution and biotransformation in roots by TEM and EDS, as well as STXM and NEXAFS. LaCl3 was also studied as a reference toxicant. La2O3 NPs and LaCl3 were both transformed to needle-like LaPO4 nanoclusters in the intercellular regions of the cucumber roots. In vitro experiments demonstrated that the dissolution of La2O3 NPs was significantly enhanced by acetic acid. Accordingly, we proposed that the dissolution of NPs at the root surface induced by the organic acids extruded from root cells played an important role in the phytotoxicity of La2O3 NPs. The reactions of active NPs at the nano-bio interface should be taken into account when studying the toxicity of dissolvable metal-based nanoparticles.

Two major types of glycosaminoglycan (GAG) polysaccharides, heparan sulfate and chondroitin sulfate, are polymerized and modified by enzymes that are encoded by more than 40 genes in animal cells. Because of the expression repertoire of the GAG assembly and modification enzymes, each heparan sulfate and chondroitin sulfate chain has a sulfation pattern, chain length, and fine structure that is potentially unique to each animal cell. GAGs interact with hundreds of proteins. Such interactions protect growth factors, chemokines, and cytokines against proteolysis. GAGs catalyze protease (such as thrombin) inhibition by serpins. GAGs regulate multiple signaling pathways including, but not limited to, fibroblast growth factor (FGF)/FGFR, hepatocyte growth factor (HGF)/c-Met, glial cell line-derived neurotrophic factor (GDNF)/c-Ret/GFRα1, vascular endothelial growth factor (VEGF)/VEGFR, platelet derived growth factor (PDGF)/PDGFR, BAFF/TACI, Indian hedgehog, Wnt, and BMP signaling pathways,where genetic studies have revealed an absolute requirement for GAGs in these pathways. Most importantly, protein/GAG aggregates induce thrombin generation and immune system upregulation by activating the contact system. Abnormal protein/GAG aggregates are associated with a variety of devastating human diseases including, but not limited to, Alzheimer's, diabetes, prion or transmissible spongiform encephalopathies, Lupus, heparin-induced thrombocytopenia/thrombosis, and different kinds of cancers. Therefore, GAGs are essential components of modern molecular biology and human physiology. Understanding GAG structure and function at molecular level with regard to development and health represents a unique opportunity in combating different kinds of human diseases.

Apoptotic cells must be rapidly eliminated to avoid harmful inflammatory and autoimmune reactions. Innate immunity is designed/poised to identify dying cells by their unique surface-associated molecular patterns. Here we demonstrate for the first time, to our knowledge, that the human complement protein properdin binds to early apoptotic T cells and initiates complement activation, leading to C3b opsonization and ingestion by phagocytic cells. Properdin binding was facilitated by the glycosaminoglycan chains of surface proteoglycans. Properdin released by activated neutrophils was particularly effective at recognition of apoptotic T cells, whereas the binding activity of properdin in the serum appeared to be inhibited. “Properdin tagging” of apoptotic T cells also induced their uptake by phagocytes independent of complement activation or other complement proteins. Although our findings were made primarily with apoptotic T cells, they suggest that properdin could play a similar role during apoptosis of other cell types.

A significant component of the innate immune system of a wide variety of animals and plants is arbitrated by cationic host defence peptides. In man, these peptides, in addition to exhibiting a direct antimicrobial activity, seems to provide a range of non-antimicrobial bioactivities related to defence, inflammation and wound healing. Despite the fact that such peptides have so far failed to reach the market, there are continued initiatives to advance such potential therapeutics to, and through, the clinic. The reasons behind such initiatives include: reduced manufacturing costs for peptides; allowing entry into therapeutic areas previously inaccessible due to cost; the continued identification of previously unknown bioactivities of such peptides; and the resurgence of interest in peptide therapeutics. As a result, clinical programmes based on cationic host defence peptides exist in the areas of infection, dermatology, cancer and inflammation. The probability of clinical success for host defence peptide-based therapeutics is on the rise as options for a wider range of clinical indications emerge.

Abstract A core–satellite‐structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica‐coated ferrite “core” and numerous “satellites” of gold nanoparticles with lots of “anchors”. The anchor, 3‐aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap‐and‐release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6 % separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N‐glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1 %) were newly identified.

We investigated occurrence of selected compounds (4-t-octylphenol: 4-t-OP; 4-nonylphenols: 4-NP; bisphenol-A: BPA; estrone: E1; 17β-estradiol: E2; triclosan: TCS) and estrogenicity in surface water and sediment of the Yellow River in China by using combined chemical analysis and in vitro yeast screen bioassay. Estrogenic compounds 4-t-OP, 4-NP, BPA, E1, E2 and TCS were measured in the water samples, with their average concentrations of 4.7, 577.9, 46.7, 1.3, ND and 6.8 ng/L, respectively. In sediment, the average concentrations of 4-t-OP, 4-NP, BPA and TCS were 35.7, 0.5, 1.7 and 0.7 ng/g while E1 and E2 were not detected in the sediments of all selected sites. In general, the estrogenic compounds in surface water and sediment of the Yellow River were at relatively low levels, thus having medium to minimal estrogenic risks in most sites except for the site of east Lanzhou with high estrogenic risks.

Cancer immunotherapy has many great achievements in recent years. One of the most promising cancer immunotherapies is PD-1/PD-L1 pathway blockade. miRNAs (MicroRNAs) belongs to small noncoding RNA and can regulate gene expression by binding to the 3′UTR. Many miRNAs can inhibit cancer growth by regulating the PD-L1 expression in cancer cells. Herein, we firstly found that PD-L1 could be the target of miR-142-5p by using bioinformatics methods, then we conduct luciferase activity assay, RT-PCR and western blot experiments to demonstrate that miR-142-5p can regulate PD-L1 expression by binding to its 3′UTR. And in vivo experiments certified that miR-142-5p overexpression can inhibit pancreatic cancer growth. Flow cytometry and RT-PCR experiment demonstrated that miR-142-5p overexpression on tumor cells inhibits the expression of PD-L1 on tumor cells which result in the increase of CD4+ T lymphocytes and CD8+ T lymphocytes, the decrease of PD-1+ T lymphocytes and increase of IFN-γ and TNF-α. So, miR-142-5p overexpression can enhance anti-tumor immunity by blocking PD-L1/PD-1 pathway. Our results identify a novel mechanism by which PD-L1 is regulated by miR-142-5p and overexpression of miR-142-5p could enhance the anti-tumor immunity.

The micro-mechanism of heavy metal cations adsorption onto biochar is critical for the renovation of heavy metal contamination. In this work, we prepared three mesoporous cellulose biochar (MCB) adsorbents with different surface area, O and N functionalities content properties through a 300 °C carbonization-KOH activation-700 °C carbonization-HNO3 oxidation process. The Cd(II) adsorption performance of three MCB was compared, the MCB-1h exhibited best Cd(II) adsorption capacity (368.8 mg/g), and even remained 89% after 5 cycles. DFT calculations identified that MCB-1h had comparatively stronger Cd(II) binding ability, and the results were well consistent with the experimental data. The adsorption micro-mechanism was analyzed in the view of classical theory and electron-scale. The classical theory results proposed that the adsorption process was dominated by chemisorption, and electron sharing or exchange between Cd(II) and biochar occurred. Electron-scale mechanism analysis found that the functional groups and aromatic could provide lone pair electrons and π electrons for the Cd(II) adsorption, respectively. The N functionalities such as amino, pyridine and pyrrole groups could raise the adsorption ability of the biochar adsorbent. Overall, our results not only provide new insights into the heavy metal adsorption, but also has significant reference value for the subsequent biochar adsorbent preparation.

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-α1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patient's fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance. Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-α1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patient's fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance.

Detecting early morphological changes in the brain and making early diagnosis are important for Alzheimer’s disease (AD). High resolution magnetic resonance imaging can be used to help diagnosis and prediction of the disease. In this paper, we proposed a machine learning method to discriminate patients with AD or mild cognitive impairment (MCI) from healthy elderly and to predict the AD conversion in MCI patients by computing and analyzing the regional morphological differences of brain between groups. Distance between each pair of subjects was quantified from a symmetric diffeomorphic registration, followed by an embedding algorithm and a learning approach for classification. The proposed method obtained accuracy of 96.5% in differentiating mild AD from healthy elderly with the whole-brain gray matter or temporal lobe as region of interest (ROI), 91.74% in differentiating progressive MCI from healthy elderly and 88.99% in classifying progressive MCI versus stable MCI with amygdala or hippocampus as ROI. This deformation-based method has made full use of the pair-wise macroscopic shape difference between groups and consequently increased the power for discrimination.

We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor.

Nanomaterial-amplified chemiluminescence (CL) has become a growing area of interest in recent years. We review the development of nanomaterial-amplified CL systems and their applications in bioassays. We mainly focus on nanoparticles (gold, platinum, silver, bimetallic, semiconductor and magnetic). Furthermore, we discuss some critical challenges in this field and possible solutions to overcome these challenges.

A seashell-like Co-MOF employing an organic-moiety-dominated intercalation/deintercalation mechanism demonstrates unprecedented long-term cyclic stability in LIBs.

Carbon superstructures with graphitized lamellar microstructures were fabricated through a modified hydrothermal method. The dual oxidation process combining acrylic acid functionalization and air thermal treatment introduced plentiful carboxyl groups on the surface of the carbon superstructures. Adsorption capacity of U(VI) on the as prepared carbon superparticles reached 197.7 mg g−1, which was 9-fold higher than that of raw glucose-derived hydrothermal carbon microspheres. The thermo-dynamic and kinetic studies demonstrated that the uptake of uranyl ions was a spontaneous and endothermic chemisorption dominated process with the abundant carboxyl groups acting as main binding sites. Impressively, the carbon superstructure adsorbents showed excellent recycling performance. To sum up, this paper proposed a novel high-performance U(VI) adsorbent from hydrothermal carbon, which exhibited potential in real application due to their outstanding reusability and scale-up capability.

Microglia are innate immune cells in the central nervous system (CNS), that supplies neurons with key factors for executing autophagosomal/lysosomal functions. Macroautophagy/autophagy is a cellular catabolic process that maintains cell balance in response to stress-related stimulation. Abnormal autophagy occurs with many pathologies, such as cancer, and autoimmune and neurodegenerative diseases. Hence, clarification of the mechanisms of autophagy regulation is of utmost importance. Recently, researchers presented microRNAs (miRNAs) as novel and potent modulators of autophagic activity. Here, we found that Mir223 deficiency significantly ameliorated CNS inflammation, demyelination and the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and increased resting microglia and autophagy in brain microglial cells. In contrast, the autophagy inhibitor 3-methylademine (3-MA) aggravated the clinical symptoms of EAE in wild-type (WT) and Mir223-deficienct mice. Furthermore, it was confirmed that Mir223 deficiency in mice increased the protein expression of ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and LC3-II in bone marrow-derived macrophage cells compared with cells from WT mice. Indeed, the cellular level of Atg16l1 was decreased in BV2 cells upon Mir223 overexpression and increased following the introduction of antagomirs. We also showed that the 3’ UTR of Atg16l1 contained functional Mir223-responsive sequences and that overexpression of ATG16L1 returned autophagy to normal levels even in the presence of Mir223 mimics. Collectively, these data indicate that Mir223 is a novel and important regulator of autophagy and that Atg16l1 is a Mir223 target in this process, which may have implications for improving our understanding of the neuroinflammatory process of EAE.Abbreviations: 3-MA: 3-methylademine; ACTB/β-actin: actin, beta; ATG: autophagy related; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CNR2: cannabinoid receptor 2 (macrophage); CNS: central nervous system; CQ: chloroquine; EAE: experimental autoimmune encephalomyelitis; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H&E: hematoxylin and eosin; ITGAM: integrin alpha M; LPS: lipoplysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; miRNAs: microRNAs; MS: multiple sclerosis; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; RA: rheumatoid arthritis; SQSTM1: sequestosome 1; TB: tuberculosis; TIMM23: translocase of inner mitochondrial membrane 23; TLR: toll-like receptor.

Abstract Purpose: To evaluate the roles of dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) and optimum tracer kinetic parameters in the noninvasive grading of the glial brain tumors with histopathological grades (I–IV). Materials and Methods: Twenty‐eight patients with histopathologically graded gliomas were imaged. Images with five flip angles were acquired before injection of gadolinium‐DTPA and were processed to calculate the T 1 value of each region of interest (ROI). All the DCE‐MRI data acquired during the injection were processed based on the MRI signal and pharmacokinetic models to establish concentration–time curves in the ROIs drawn within the tumors, contralateral normal areas, and area of the individual artery input functions (iAIF) of each patient. A nonlinear least‐square‐fitting method was used to obtain tracer kinetic parameters. Kruskal–Wallis H ‐test and Mann–Whitney U ‐test were applied to these parameters in different histopathological grade groups for statistical differences ( P &lt; 0.05). Results: Volume transfer coefficient (K trans ) and extravascular extracellular space volume fraction (V e ) calculated using iAIFs can be used not only to distinguish the low (ie, I and II) from the high (ie, III and IV) grade gliomas (P( ) &lt; 0.001 and P Ve &lt; 0.001), but also grade II from III (P( ) = 0.016 and P Ve = 0.033). Conclusion: K trans is the most sensitive and specific parameter in noninvasive grading, distinguishing the high (III and IV) from the low (I and II) grade and high grade III from low grade II gliomas. J. Magn. Reson. Imaging 2012;36:355–363. © 2012 Wiley Periodicals, Inc.

Abstract Poria cocos is an edible medicinal fungus known as “Fuling” in Chinese and has been used as a Chinese traditional medicine for more than two thousand years. Pharmacological studies reveal that polysaccharide is the most abundant substance in Poria cocos and has a wide range of biological activities including antitumour, immunomodulation, anti‐inflammation, antioxidation, anti‐ageing, antihepatitis, antidiabetics and anti‐haemorrhagic fever effects. As a result, “ Poria cocos polysaccharide oral solution” was developed and sold as an over‐the‐counter health supplement since 1970s. In 2015, “Polysaccharidum of Poria cocos oral solution” was approved as a drug by Chinese Food and Drug Administration for treating multiple types of cancers, hepatitis and other diseases alone or during chemo‐ or radiation therapy for patients with cancer. In this article, biochemical, preclinical and clinical studies of Poria cocos polysaccharide from 72 independent studies during the past 46 years (1970‐2016) based on PubMed, VIP (Chongqing VIP Chinese Scientific Journals Database), CNKI (China National Knowledge Infrastructure) and Wanfang database searches are summarized. The structure, pharmacological effects, clinical efficacy, immunobalancing molecular mechanism and toxicity of Poria cocos polysaccharide are deliberated to provide a general picture of Poria cocos polysaccharide as a clinically used antitumour drug.

Abstract The crystal phase plays an important role in controlling the properties of a nanomaterial; however, it is a great challenge to obtain a nanomaterial with high purity of the metastable phase. For instance, the large‐scale synthesis of the metallic phase MoS 2 (1T‐MoS 2 ) is important for enhancing electrocatalytic reaction, but it can only be obtained under harsh conditions. Herein, a spatially confined template method is proposed to synthesize high phase‐purity MoS 2 with a 1T content of 83 %. Moreover, both the confined space and the structure of template will affect the purity of 1T‐MoS 2 ; in this case, this approach was extended to other similar spatially confined templates to obtain the high‐purity material. The obtained ultrathin nanosheets exhibit good electrocatalytic activity and excellent stability in the hydrogen evolution reaction.

The present study reports a straightforward template-free route for the synthesis of core–shell Co@CoO nanocomposites by the controlled reduction of Co3O4 nanospheres. The target Co@CoO nanoparticles consist of an unsealed hollow porous CoO shell with a metal Co core, in which the outer porous CoO shell as the active anode material can be fully in contact with the electrolyte. The void within the particles provides a remarkable buffer to tolerate volume changes of the electrode materials during the insertion and extraction of lithium. Most importantly, the inner nanosized metal Co core gives a new impetus to the reversible decomposition of Li2O due to its catalytic activity. Furthermore, the exposed metal Co portion outside the nanoshells provides a favorable electrical contact between adjacent particles and greatly improves the efficiency of the electronic connection between the active material and the current collector. The Co@CoO nanocomposite maintains an excellent reversible capacity over 800 mA h g−1 after 50 cycles with an initial coulombic efficiency of 74.2%, which is much higher than that of pure CoO (67.8%). This superior electrochemical performance is closely related to the unique composition and nanostructure of the electrode material. Notably, it is the first case of a hybrid-structured Co@CoO anode material derived from the reduction process from oxide precursors. Such a conclusion may be advantageously used to guide the design of a wide range of nanostructured metal oxides.

Recycled coarse aggregate (RCA) concrete has attracted more and more attention worldwide in recent years due to the exhaustion of natural coarse aggregate and environmental pollution from construction and demolition waste in civil engineering. In this study, experiments were carried out on over 100 specimens to investigate the mechanical properties and failure mode of concrete with different volume content of steel fibers (0%, 0.5%, 1%, 1.5%, and 2%) and different RCA replacement ratio (0%, 30%, 50% and 100%) under direct shear load. The results show that addition of steel fibers can effectively improve the shear strength and shear toughness of RCA concrete. For a given compressive strength, the RCA replacement ratio has negligible impact on shear strength, but shear deformation and toughness increase as RCA replacement ratio reaches above a ‘limiting value’. A shear strength formula for steel fiber reinforced RCA concrete (SFRCAC) based on compressive strength and characteristic coefficient of steel fiber has been put forward.

This paper presents the experimental results on the flexural performance of steel fiber reinforced recycled coarse aggregate concrete (SFRCAC). Test parameters include compressive strength (30 MPa, 45 MPa, 60 MPa), recycled coarse aggregate (RCA) replacement ratio (0, 30%, 50%, 100%), and steel fiber volume fraction (0, 0.5%, 1%, 1.5%, 2%). Evaluation method of flexural performance in ASTM C1609 and Chinese Standard JG/T 427-2015 was investigated with respect to the variance in test parameters. Test results showed that compressive strength has the similar influence on SFRCAC and normal concrete; with RCA replacement ratio increasing, the flexural performance of SFRCAC before crack is quite similar, flexural strength and flexural toughness increase slightly, deflection increases heavily; with the steel fiber volume fraction (Vf) increasing, the reinforcement effect is not obvious when Vf < 0.5%, improved significantly when Vf increases from 0.5% to 1%, growth trend becomes flatter when Vf is above 1%. The evaluation methods of flexural performance in ASTM C1609 and JG/T 427-2015 are not suitable for SFRCAC due to its deflection is much higher than normal concrete. A new set of evaluation indexes have been put forward and test results have demonstrated that this evaluation method is concise and effective.

The aim of this study was to determine the effects of psychological interventions (e.g. cognitive restructuring, relaxation) on physiological and psychological health in osteoarthritis patients. A systematic literature search was done using PubMed, Embase, PsycINFO, Web of Science, China National Knowledge Infrastructure, and Wanfang Database through November 2016. Studies were included if they used a randomized controlled trial designed to explore the effects of psychological interventions in osteoarthritis patients. Two independent authors assessed the methodological quality of the trials using criteria outlined by Jadad et al. Meta-analysis was done with the Revman5.0. Twelve randomized controlled trials, including 1307 osteoarthritis patients, met the study inclusion criteria. Meta-analysis showed that psychological interventions could reduce the levels of pain [standard mean difference (SMD) −0.28, 95% CI −0.48, −0.08, P-value 0.005)] and fatigue (SMD −0.18, 95% CI −0.34, −0.01, P-value 0.04). In addition, psychological interventions significantly improved osteoarthritis patients’ self-efficacy (SMD 0.58, 95% CI 0.40, 0.75, P-value 0.00) and pain coping (MD 1.64, 95% CI 0.03, 3.25, P-value 0.05). Although the effects on physical function, anxiety, depression, psychological disability were in the expected direction, they were not statistically significant. In conclusion, the role of psychological interventions in the management of osteoarthritis remains equivocal. Some encouraging results were seen with regard to pain, pain coping, self-efficacy, and fatigue. We believe that more methodologically rigorous large-scale randomized controlled trials are necessary to answer this study question.