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Chilling causes a significant decline in photosynthesis in tomato plants. Tomato tryptophan decarboxylase gene 1 (SlTDC1) is the first rate-limiting gene for melatonin (MT) biosynthesis and is involved in the regulation of photosynthesis under various abiotic stresses. However, it is not clear whether SlTDC1 participates in the photosynthesis of tomato under chilling stress. Here, we obtained SlTDC1 overexpression transgenic tomato seedlings, which showed higher SlTDC1 mRNA abundance and MT content compared with the wild type (WT). The results showed that the overexpression of SlTDC1 obviously alleviated the chilling damage to seedlings in terms of the lower electrolyte leakage rate and hydrogen peroxide content, compared with the WT after 2 d of chilling stress. Moreover, the overexpression of SlTDC1 notably increased photosynthesis under chilling stress, which was related to the higher chlorophyll content, normal chloroplast structure, and higher mRNA abundance and protein level of Rubisco and RCA, as well as the higher carbon metabolic capacity, compared to the WT. In addition, we found that SlTDC1-overexpressing seedlings showed higher Wk (damage degree of OEC on the PSII donor side), φEo (quantum yield for electron transport in the PSII reaction center), and PIABS (photosynthetic performance index) than WT seedlings after low-temperature stress, implying that the overexpression of SlTDC1 decreased the damage to the reaction center and donor-side and receptor-side electron transport of PSII and promoted PSI activity, as well as energy absorption and distribution, to relieve the photoinhibition induced by chilling stress. Our results support the notion that SlTDC1 plays a vital role in the regulation of photosynthesis under chilling stress.

Due to the great potential of surface-enhanced Raman scattering (SERS) as local vibrational probe of lipid-nanostructure interaction in lipid bilayers, it is important to characterize these interactions in detail. The interpretation of SERS data of lipids in living cells requires an understanding of how the molecules interact with gold nanostructures and how intermolecular interactions influence the proximity and contact between lipids and nanoparticles. Ceramide, a sphingolipid that acts as important structural component and regulator of biological function, therefore of interest to probing, lacks a phosphocholine head group that is common to many lipids used in liposome models. SERS spectra of liposomes of a mixture of ceramide, phosphatidic acid, and phosphatidylcholine, as well as of pure ceramide and of the phospholipid mixture are reported. Distinct groups of SERS spectra represent varied contributions of the choline, sphingosine, and phosphate head groups and the structures of the acyl chains. Spectral bands related to the state of order of the membrane and moreover to the amide function of the sphingosine head groups indicate that the gold nanoparticles interact with molecules involved in different intermolecular relations. While cryogenic electron microscopy shows the formation of bilayer liposomes in all preparations, pure ceramide was found to also form supramolecular, concentric stacked and densely packed lamellar, nonliposomal structures. That the formation of such supramolecular assemblies supports the intermolecular interactions of ceramide is indicated by the SERS data. The unique spectral features that are assigned to the ceramide-containing lipid model systems here enable an identification of these molecules in biological systems and allow us to obtain information on their structure and interaction by SERS.

Grafting is widely applied to enhance the tolerance of some vegetables to biotic and abiotic stress. Salicylic acid (SA) is known to be involved in grafting-induced chilling tolerance in cucumber. Here, we revealed that grafting with pumpkin (Cucurbita moschata, Cm) as a rootstock improved chilling tolerance and increased the accumulation of SA, abscisic acid (ABA) and hydrogen peroxide (H2O2) in grafted cucumber (Cucumis sativus/Cucurbita moschata, Cs/Cm) leaves. Exogenous SA improved the chilling tolerance and increased the accumulation of ABA and H2O2 and the mRNA abundances of CBF1, COR47, NCED, and RBOH1. However, 2-aminoindan-2-phosphonic acid (AIP) and L-a-aminooxy-b-phenylpropionic acid (AOPP) (biosynthesis inhibitors of SA) reduced grafting-induced chilling tolerance, as well as the synthesis of ABA and H2O2, in cucumber leaves. ABA significantly increased endogenous H2O2 production and the resistance to chilling stress, as proven by the lower electrolyte leakage (EL) and chilling injury index (CI). However, application of the ABA biosynthesis inhibitors sodium tungstate (Na2WO4) and fluridone (Flu) abolished grafting or SA-induced H2O2 accumulation and chilling tolerance. SA-induced plant response to chilling stress was also eliminated by N,N′-dimethylthiourea (DMTU, an H2O2 scavenger). In addition, ABA-induced chilling tolerance was attenuated by DMTU and diphenyleneiodonium (DPI, an H2O2 inhibitor) chloride, but AIP and AOPP had little effect on the ABA-induced mitigation of chilling stress. Na2WO4 and Flu diminished grafting- or SA-induced H2O2 biosynthesis, but DMTU and DPI did not affect ABA production induced by SA under chilling stress. These results suggest that SA participated in grafting-induced chilling tolerance by stimulating the biosynthesis of ABA and H2O2. H2O2, as a downstream signaler of ABA, mediates SA-induced chilling tolerance in grafted cucumber plants.

Monolayer-ordered gold nanoring arrays were prepared by ion-sputtering method and used as surface enhanced Raman spectroscopy (SERS) substrates to test the individual atmospheric aerosols particle. Compared to other methods used for testing atmospheric aerosols particles, the collection and subsequent detection in our work is performed directly on the gold nanoring SERS substrate without any treatment of the analyte. The SERS performance can be tuned by changing the depth of the gold nanoring cavity as originating from coupling of dipolar modes at the inner and outer surfaces of the nanorings. The electric field exhibits uniform enhancement and polarization in the ordered Au nanoring substrate, which can improve the accuracy for detecting atmospheric aerosol particles. Combined with Raman mapping, the information about chemical composition of individual atmospheric aerosols particle and distribution of specific components can be presented visually. The results show the potential of SERS in enabling improved analysis of aerosol particle chemical composition, mixing state, and other related physicochemical properties.

Water-soluble organic carbon (WSOC), which is closely related to biogenic emissions, is of great importance in the atmosphere for its ubiquitous existence and rich abundance. Levoglucosan, a typical WSOC, is usually considered to be stable and thus used as a tracer of biomass burning. However, we found that levoglucosan can be photo-oxidized on mineral dust, with formic acid, oxalic acid, glyoxylic acid, 2,3-dioxopropanoic acid, dicarbonic acid, performic acid, mesoxalaldehyde, 2-hydroxymalonaldehyde, carbonic formic anhydride, and 1,3-dioxolane-2,4-dione detected as main products. Further, we observed the heterogeneous uptake of NH3 promoted by the carboxylic acids stemming from the photocatalytic oxidation (PCO) of levoglucosan. The mineral-dust-initiated PCO of levoglucosan and enhanced heterogeneous uptake of NH3, which are highly influenced by irradiation and moisture conditions, were for the first time revealed. The reaction mechanisms and pathways were studied in detail by diffuse reflection infrared Fourier transform spectroscopy (DRIFTS), high-pressure photon ionization time-of-flight mass spectrometry (HPPI-ToF-MS) and flow reactor systems. Diverse WSOC constituents were studied as well, and the reactivity toward NH3 is related to the number of hydroxyl groups of the WSOC molecules. This work reveals a new precursor of secondary organic aerosols and provides experimental evidence of the existence of organic ammonium salts in atmospheric particles.

Detecting atmospheric bioaerosols in a quantitative way is highly desirable for public health and safety. This work demonstrates that surface-enhanced Raman spectroscopy (SERS) is a simple and rapid analytical technique for the detection of atmospheric bioaerosols, on a Klarite substrate. For both simulated and ambient bioaerosols, this detection assay results in an increase in the enhancement factor of the Raman signal. We report a strong SERS signal generated by bioaerosols containing living Escherichia coli deposited on Klarite. Furthermore, we demonstrate that SERS mapping can be used to estimate the percentage of airborne, living Escherichia coli. Moreover, Klarite provides differently distinct SERS spectra at different bacterial growth phases, indicating its potential to identify changes occurring in the bacterial envelope. Finally, we applied SERS for the rapid detection of Escherichia coli in ambient bioaerosols without using time-consuming and laborious culture processes. Our results represent rapid, culture-free and label-free detection of airborne bacteria in the real-world environment.

Surface enhanced Raman spectroscopy (SERS) shows great promise in studying individual atmospheric aerosol. However, the lack of efficient, stable, uniform, large-array, and low-cost SERS substrates constitutes a major roadblock. Herein, a new SERS substrate is proposed for detecting individual atmospheric aerosol particles. It is based on the sphere segment void (SSV) structure of copper and silver (Cu/Ag) alloy. The SSV structure is prepared by an electrodeposition method and presents a uniform distribution, over large 2 cm 2 arrays and at low cost. The substrate offers a high SERS enhancement factor (due to Ag) combined with lasting stability (due to Cu). The SSV structure of the arrays generates a high density of SERS hotspots (1.3 × 10 14/cm 2), making it an excellent substrate for atmospheric aerosol detection. For stimulated sulfate aerosols, the Raman signal is greatly enhanced (>50 times), an order of magnitude more than previously reported substrates for the same purpose. For ambient particles, collected and studied on a heavy haze day, the enhanced Raman signal allows ready observation of morphology and identification of chemical components, such as nitrates and sulfates. This work provides an efficient strategy for developing SERS substrate for detecting individual atmospheric aerosol.

SO2 and CO2 emitted from using of fossil fuels are causing serious environmental issues. Herein we put forward a promising strategy to realize CO2 reduction and SO2 removal simultaneously through a photoelectrochemical (PEC) process based on nanoelectrodes. In the present work, SO2 was oxidized to SO42− on BiVO4 photoanode after being absorbed by NaHCO3 solutions. At the same time, CO2 reduction occurred on nanoflower-like copper-indium alloy cathode. The absorption efficiency of SO2 was greater than 95%, and SO32− was oxidized to SO42− with oxidation efficiency about 50% in 3 h. Moreover, the energy of SO2 was recycled to assist the CO2 reduction on the cathode, consequently, the photocurrent densities were increased at least 3 times after introducing SO2. CO2 reduction with CO and HCOOH as main products was improved significantly when SO2 removal was conducted simultaneously. In-situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) experiments were also performed to study the adsorption and conversion of SO2 on BiVO4 nanoparticles. The proposed approach could efficiently remove SO2 and recycle the energy of it to reduce CO2 into useful chemicals. This work provides a sustainable strategy for dealing with CO2 and SO2 in the flue gas by combining desulfurization and CO2 reduction in one PEC cell using nanoelectrodes.

Sulfite is recently found to be promising in enhancing photocatalytic pollutants degradation, and it is a byproduct from flue gas desulfuration process. Herein, 4-chlorophenol (4-CP) photodegradation was systematically investigated in a sulfite mediated system with g-C3N4 as photocatalyst. The degradation efficacy was improved by about 3 times with addition of 25 mM Na2SO3. The dominant responsible reactive oxygen species for chlorophenols remediation in the presence of sulfite included O2·-, SO3·-, and SO4·- as confirmed by radical quenching experiments and electron spin resonances technology. In-situ DRIFTs results indicated the improved cleavage of C-Cl and C-H bonds with the simultaneous formation of C O and C C bonds when bisulfite was added. Degradation intermediates such as 4-chlorocatechol, hydroquinone, and muconic acid were detected by HPLC-MS. Furthermore, the photodegradation mechanisms were discussed in the presence of sulfite. Other chlorophenols (phenol, 2-CP, 2,4-DCP, and their mixture) were also efficiently removed in the system, suggesting that sulfite could be universally applied in photocatalytic wastewater purification.

Microdroplets present unique physiochemical properties, which can influence chemical reactions and atmospheric aerosol processes. The microdroplets’ pH is one of the most important factors that dictate chemical processes, such as heterogeneous and condensed reactions. However, currently, there is no well-established method for directly measuring the pH in microdroplets. Herein, we develop a straightforward method, for the first time, to directly measure the pH of size-resolved, micron-sized single droplets with Surface Enhanced Raman Scattering (SERS)-activated pH-indicator paper; realized by embedded Au nanoparticles (20, 40 and 60 nm). We establish the key parameters for optimizing the method, and demonstrate its accuracy by benchmarking various acids and solutions with different ionic strength. With the established method, we report the pH dependence on the size of microdroplets. Microdroplets of sizes down to 230 nm can be analyzed by our method. We also showcase our method’s applicability to ambient samples, establishing its promising potential beyond the laboratory environment.

A highly efficient diversified methodology for preparation of benzimidazole, quinazolin-4(3H)-ones and 1,4-benzodiazepine-2,5-diones is established using a palladium-catalyzed transfer hydrogenation (CTH)/condensation cascade of o-nitroaniline and o-nitrobenzamides in a triethylamine–formic acid azeotropic mixture (2:5) under microwave irradiation.

Studies on the further development of the sequential glycosylations of the vancomycin aglycon catalyzed by the glycosyltransferases GtfE and GtfD and the observation of unusual, perhaps unexpected, aglycon substrate substituent effects on the rate and efficiency of the initial glycosylation reaction are reported.

The mannopeptimycins are a class of glycopeptide natural products with unusual structures and potent antibiotic activity against a range of Gram-positive multidrug-resistant bacteria. Their cyclic hexapeptide core features a pair of unprecedented β-hydroxyenduracididines (L- and D-βhEnd), an O-glycosylated D-Tyr carrying an α-linked dimannose, and a β-methylated Phe residue. The D-βhEnd unit also carries an α-linked mannopyranose at the most hindered N of its cyclic guanidine ring. Herein, we report the first total synthesis of mannopeptimycin α and β with fully elaborated N- and O-linked sugars. Critically, a gold-catalyzed N-glycosylation of a D-βhEnd substrate with a mannosyl ortho-alkynylbenzoate donor enabled the synthesis of the most challenging N-Man-D-βhEnd unit with excellent efficiency and stereoselectivity. The L-βMePhe unit was prepared using a Pd-catalyzed C-H arylation method. The L-βhEnd, D-Tyr(di-Man), and L-βMePhe units were prepared in gram quantities. A convergent assembly of the cyclic peptide scaffold and a single global hydrogenolysis deprotection operation provided mannopeptimycin α and β.

Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.

Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with diverse human germline framework regions (FRs), which were selected based on structural and sequence similarities between mouse variable domains and a repertoire of human antibody germline genes. SDRO consisted of diversifying specificity-determining residues and selecting variants with improved affinity using phage display. HFS of m836 resulted in a 5-fold loss of affinity, whereas SDRO increased the affinity up to 100-fold compared to the HFS antibody. Crystal structures of Fabs in complex with IL-13 were obtained for m836, the HFS variant chosen for SDRO, and one of the highest-affinity SDRO variants. Analysis of the structures revealed that major conformational changes in FR-H1 and FR-H3 occurred after FR replacement, but none of them had an evident direct impact on residues in contact with IL-13. Instead, subtle changes affected the V L /V H (variable-light domain/variable-heavy domain) interface and were likely responsible for the 5-fold decreased affinity. After SDRO, increased affinity resulted mainly from rearrangements in hydrogen-bonding pattern at the antibody/antigen interface. Comparison with m836 putative germline genes suggested interesting analogies between natural affinity maturation and the engineering process that led to the potent HFA anti-human IL-13 antibody.

Despite of the medicinal significance of benzoxaboroles, with the newly discovered clinical compound AN2690 as an example, the synthetic method for rapid diversification of this novel scaffold is lacking. To this end, a versatile and scalable synthesis of formyl-substituted benzoxaboroles is described here. A key step is the mono-oxidation of the two hydroxyls in compound 4 by taking advantage of the stable oxaborole ring in non-coordinating solvents, which was devised based on the study of the intramolecular coordination and exchange properties.

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.

Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.