Amino-Acid-Induced Circular Polarized Luminescence in One-Dimensional Manganese(II) Halide Hybrid.

Circular polarized luminescence (CPL)-active materials attract great attentions owing to their widely applications in 3D optical displays and encrypted transmission. Inspired by the strategies adopted in perovskite based CPL materials, herein, CPL-active hybrids (D)- and (L)-(tert-butyl prolinate)MnCl3 were successfully prepared by assembling chiral D/L tert-butyl prolinate with manganese (II) chloride. Single crystal structures show the as-formed hybrids possess one-dimensional (1D) structure containing linear chains of face-sharing MnCl6 octahedral surrounded by prolinate cations. The 1D Mn(II) hybrids display strong red emission peaked at 646 nm with PLQY of 67.1 % and 57.2 % for d-type and l-type, respectively, representing the highest PLQY for 1D MnII hybrids. Interestingly, the 1D Mn(II) hybrids exhibit prominent circular dichroism (CD) signals and remarkable CPL activity with the dissymmetry factor g of 6.1*10-3 and -6.3*10-3 from 550 to 800 nm for (D)- and (L)-(tert-butyl prolinate)MnCl3 , respectively, owing to the existence of chiral cations. It is worthy noted the obtained g represents the highest value for non-lead organic-inorganic hybrids.

Construction and characterization of the interdomain chimeras using Cry11Aa and Cry11Ba from Bacillus thuringiensis and identification of a possible novel toxic chimera.

Three structural domains of mosquitocidal Cry11Aa and Cry11Ba from Bacillus thuringiensis were exchanged to produce interdomain chimeras [BAA (11Ba/11Aa/11Aa), ABA (11Aa/11Ba/11Aa), AAB (11Aa/11Aa/11Ba), ABB (11Aa/11Ba/11Ba), BAB (11Ba/11Aa/11Ba), BBA (11Ba/11Ba/11Aa]. Chimeras BAB, BAA, BBA, and AAB formed inclusion bodies in the crystal-negative B. thuringiensis host and produced expected protein bands on SDS-PAGE gel. However, no inclusion body or target protein could be found for chimeras ABA and ABB. In bioassays using the fourth-instar larvae of Culex quinquefasciatus and Aedes aegypti, AAB had ~50 % lethal concentrations of 4.8 and 2.2 µg ml(-1), respectively; however, the rest of chimeras were not toxic. This study thus helps to understand the domain-function relationships of the Cry11Aa and Cry11Ba toxins. The toxic chimera, AAB, might be a candidate for mosquito control as its amino acid sequence is different from the two parental toxins.

Nanoscale mono- and multi-layer cylinder structures formed by recombinant S-layer proteins of mosquitocidal Bacillus sphaericus C3-41.

The mature surface layer (S-layer) protein SlpC of mosquitocidal Bacillus sphaericus C3-41 comprises amino acids 31-1,176 and could recrystallize in vitro. The N-terminal SLH domain is responsible for binding function. Deletion of this part, S-layer proteins could not bind to the cell wall sacculi. To investigate the self-assembly ability of SlpC from B. sphaericus, nine truncations were constructed and their self-assembly properties were compared with the recombinant mature S-layer protein rSlpC31₋1,176. The results showed that rSbsC31₋1,176 and truncations rSlpC211₋1,176, rSlpC278₋1,176, rSlpC31₋1,100, and rSlpC31₋1,050 could assemble into multilayer cylinder structures, while N-terminal truncations rSlpC338₋1,176, rSlpC438₋1,176, and rSlpC498₋1,176 mainly showed monolayer cylinders in recombinant Escherichia coli BL21 (DE3) cells. Growth phase analysis of the self-assembly process revealed that rSlpC498₋1,176 mainly formed monolayer cylinders in the early stage (0.5 and 1 h induction of expression), but few double-layer or multilayer cylinders were also found with the cells growing, while rSlpC31₋1,176 could formed multilayer cylinders in all the growth stage in the E. coli cells. It is concluded that the deletion of the C-terminal 126 aa or the N-terminal 497 aa did not interfere with the self-assembly process, the fragment (amino acids 278 to 337) is essential for the multilayer cylinder formation in E. coli BL21 (DE3) cells in the early stage and the fragment (amino acids 338 to 497) is related to monolayer cylinder formation. The information is important for further studies on the assembly mechanism of S-layer proteins and forms a basis for further studies concerning surface display and nanobiotechnology.

Ultrasmall PtAu2 nanoclusters activate endogenous anti-inflammatory and anti-oxidative systems to prevent inflammatory osteolysis.

Rationale: Inflammatory osteolysis, characterized by abundant immune cell infiltration and osteoclast (OC) formation, is a common complication induced by bacterial products and/or wear particles at the bone-prosthesis interface that severely reduces long-term stability after implantation. Molecular nanoclusters are ultrasmall particles with unique physicochemical and biological properties that have great potential as theranostic agents for treating inflammatory diseases. Methods: In this study, heterometallic PtAu2 nanoclusters with sensitive nitric oxide-responsive phosphorescence turn-on characteristics and strong binding interactions with cysteine were designed, making them desirable candidates for the treatment of inflammatory osteolysis. Results: PtAu2 clusters exhibited satisfactory biocompatibility and cellular uptake behavior, with potent anti-inflammatory and anti-OC activities in vitro. In addition, PtAu2 clusters alleviated lipopolysaccharide-induced calvarial osteolysis in vivo and activated nuclear factor erythroid 2-related factor 2 (Nrf2) expression by disrupting its association with Kelch-like ECH-associated protein 1 (Keap1), thereby upregulating the expression of endogenous anti-inflammatory and anti-oxidative products. Conclusion: Through the rational design of novel heterometallic nanoclusters that activate the endogenous anti-inflammatory system, this study provides new insights into the development of multifunctional molecular therapeutic agents for inflammatory osteolysis and other inflammatory diseases.

CcpA-mediated enhancement of sugar and amino acid metabolism in Lysinibacillus sphaericus by NMR-based metabolomics.

Lysinibacillus sphaericus is a bacterium incapable of metabolizing sugars with the sole exception of N-acetylglucosamine. To unravel the regulatory role of catabolite control protein A (CcpA) in the sugar metabolism of L. sphaericus, a ccpA deficient mutant was constructed by homologous recombination. The mutant showed growth deficiency and a low efficiency of carbon and energy utilization. NMR spectroscopy in combination with multivariate data analysis revealed that the metabolome of L. sphaericus was dominated by 25 metabolites mainly including amino acids, carbohydrate derivatives and organic acids, and that the mutation of the ccpA gene caused significant reduction of leucine, valine, alanine, threonine, glutamate, lysine, d-ornithine, tyrosine, uridine 5'-diphospho-N-acetlyglucosamine formate, fumarate, phenylalanine, aspartate, asparagine, and acetate but elevation of ribose-5-phosphate, and uracil. Furthermore, the networks of CcpA-mediated regulation based on the metabolome were constructed by arrangement of significantly decreasing or increasing metabolites. The network map suggests CcpA regulates and promotes sugar and amino acid metabolism of L. sphaericus.

Comparison of humoral immune responses to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus using a viral proteome microarray.

BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus, and Kaposi's sarcoma-associated herpesvirus (KSHV) has a restricted seroprevalence. Both viruses are associated with malignancies that have an increased frequency in individuals who are coinfected with human immunodeficiency virus type 1 (HIV-1). METHODS: To obtain an overview of humoral immune responses to these viruses, we generated a protein array that displayed 174 EBV and KSHV polypeptides purified from yeast. Antibody responses to EBV and KSHV were examined in plasma from healthy volunteers and patients with B cell lymphoma or with AIDS-related Kaposi's sarcoma or lymphoma. RESULTS: In addition to the commonly studied antigens, IgG responses were frequently detected to the tegument proteins KSHV ORF38 and EBV BBRF and BGLF2 and BNRF1 and to the EBV early lytic proteins BRRF1 and BORF2. The EBV vIL-10 protein was particularly well recognized by plasma IgA. The most intense IgG responses to EBV antigens occurred in HIV-1-positive patients. No clear correlation was observed between viral DNA load in plasma and antibody profile. CONCLUSIONS: The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

Highly Phosphorescent Dimers of PtAu2 Complexes and the Use in Solution-Processed OLEDs.

Two asymmetric PtAu2 complexes having HC≡CC6H4C≡CH (1,4-diethynylbenzene) or HC≡CCarbC≡CH (2,7-diethynyl-9-(2,3,5,6-tetrafluorophenyl)-9H-carbazole) and the corresponding bis(acetylide)-linked Pt2Au4 complexes are prepared and characterized. The structures of PtAu2 complexes 1 and 3 together with Pt2Au4 complex 2 are determined by X-ray crystallography. Relative to PtAu2 complexes, bis(acetylide)-linked Pt2Au4 complexes not only display a distinct red shift of the emission but also provide a much higher phosphorescent efficiency. Utilizing highly emissive Pt2Au4 complexes as phosphorescent dopants, high-efficiency solution-processed OLEDs are obtained with peak current efficiency of 75.9 cd A-1 and external quantum efficiency of 19.0% at luminance of 336 cd m-2 and voltage of 5.2 V. When two PtAu2 moieties are linked by a bis(acetylide) ligand, the corresponding Pt2Au4 complexes show a much improved electroluminescent performance compared with that of asymmetric PtAu2 complexes.

Allelic diversity and population structure of Bacillus sphaericus as revealed by multilocus sequence typing.

The genetic diversity of 35 Bacillus sphaericus strains was analyzed by a newly developed multilocus sequence typing (MLST) scheme, toxin gene pool survey, and mosquito bioassay. The results demonstrated that strains assigned to the same sequence type (ST) had the same occurrence of toxin genes. Further sequence analysis revealed that toxic strains presented a nearly clonal population structure, whereas nontoxic strains had a high level of heterogeneity and were significantly distinct from toxic strains.

A dual-response fluoran-phenothiazine hybrid fluorescent probe for selective sensing of Fe3+ and ClO- and cell imaging application.

Bifunctional fluorescent probes with dual-emission response attract extensive attention. A novel fluorescent probe FP, a hybrid of fluoran and phenothiazine, has been designed and synthesized for selective sensing of Fe3+ and ClO- with dual-emission changes, which involes mechanisms of Fe3+-promoted spirolactone ring opening and ClO--induced oxidation of phenothiazine moiety, respectively. In addition, the detection limits for Fe3+ and ClO- were estimated to be 49.1 and 35.9 nM, respectively. Significantly, FP can be employed as an tracer for the detection of Fe3+ ions within living HeLa cells by fluorescence imaging.

Development and Molecular Cytogenetic Identification of a New Wheat-Psathyrostachys huashanica Keng Translocation Line Resistant to Powdery Mildew.

Psathyrostachys huashanica Keng, a wild relative of common wheat with many desirable traits, is an invaluable source of genetic material for wheat improvement. Few wheat-P. huashanica translocation lines resistant to powdery mildew have been reported. In this study, a wheat-P. huashanica line, E24-3-1-6-2-1, was generated via distant hybridization, ethyl methanesulfonate (EMS) mutagenesis, and backcross breeding. A chromosome karyotype of 2n = 44 was observed at the mitotic stage in E24-3-1-6-2-1. Genomic in situ hybridization (GISH) analysis revealed four translocated chromosomes in E24-3-1-6-2-1, and P. huashanica chromosome-specific marker analysis showed that the alien chromosome fragment was from the P. huashanica 4Ns chromosome. Moreover, fluorescence in situ hybridization (FISH) analysis demonstrated that reciprocal translocation had occurred between the P. huashanica 4Ns chromosome and the wheat 3D chromosome; thus, E24-3-1-6-2-1 carried two translocations: T3DS·3DL-4NsL and T3DL-4NsS. Translocation also occurred between wheat chromosomes 2A and 4A. At the adult stage, E24-3-1-6-2-1 was highly resistant to powdery mildew, caused by prevalent pathotypes in China. Further, the spike length, numbers of fertile spikelets, kernels per spike, thousand-kernel weight, and grain yield of E24-3-1-6-2-1 were significantly higher than those of its wheat parent 7182 and addition line 24-6-3-1. Thus, this translocation line that is highly resistant to powdery mildew and has excellent agronomic traits can be used as a novel promising germplasm for breeding resistant and high-yielding cultivars.

Conjugative transfer of insecticidal plasmid pHT73 from Bacillus thuringiensis to B. anthracis and compatibility of this plasmid with pXO1 and pXO2.

Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1(+) pXO2(-)) and Ba63605R (pXO1(-) pXO2(+)), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 x 10(-4) and 1.6 x 10(-4) CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment.

From homonuclear to heteronuclear: a viable strategy to promote and modulate phosphorescence.

Numerous mononuclear platinum(ii) complexes are non-emissive or weakly emissive under ambient conditions, but the corresponding Pt-M (M = Cu(i), Ag(i), Au(i), etc.) heteronuclear assemblies could become intensely luminescent because of the inhibition of non-radiative relaxation and the promotion of intersystem crossing from singlet to triplet state through Pt-M intermetallic interactions. To this end, the fabrication of specifically structured Pt-M complexes by the use of slightly luminescent homonuclear Pt(ii) precursors provides a promising approach to switching on phosphorescence as well as modulating emission energy and colour. This feature article is aimed at providing some typical examples for attaining highly phosphorescent Pt-M heteronuclear complexes using homonuclear Pt(ii) precursors, focusing on the assembly strategy, the correlation of emissive properties to the structures, and the application of phosphorescence in sensing and light-emitting devices.

Complete genome sequence of the mosquitocidal bacterium Bacillus sphaericus C3-41 and comparison with those of closely related Bacillus species.

Bacillus sphaericus strain C3-41 is an aerobic, mesophilic, spore-forming bacterium that has been used with great success in mosquito control programs worldwide. Genome sequencing revealed that the complete genome of this entomopathogenic bacterium is composed of a chromosomal replicon of 4,639,821 bp and a plasmid replicon of 177,642 bp, containing 4,786 and 186 potential protein-coding sequences, respectively. Comparison of the genome with other published sequences indicated that the B. sphaericus C3-41 chromosome is most similar to that of Bacillus sp. strain NRRL B-14905, a marine species that, like B. sphaericus, is unable to metabolize polysaccharides. The lack of key enzymes and sugar transport systems in the two bacteria appears to be the main reason for this inability, and the abundance of proteolytic enzymes and transport systems may endow these bacteria with exclusive metabolic pathways for a wide variety of organic compounds and amino acids. The genes shared between B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, including mobile genetic elements, membrane-associated proteins, and transport systems, demonstrated that these two species are a biologically and phylogenetically divergent group. Knowledge of the genome sequence of B. sphaericus C3-41 thus increases our understanding of the bacilli and may also offer prospects for future genetic improvement of this important biological control agent.

[Expression of mosquitocidal Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus].

Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al. , 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing CytlAa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and CytlAa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid.

Substrate preference of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase in Burkholderia thailandensis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and AdoHcyase from Burkholderia thailandensis were cloned and characterized. The K(m) values of MTAN for 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) were 19 and 58 µM, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B. thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, AdoHcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the K(m) value of AdoHcyase for SAH was determined to be 40 µM. Sequence analysis revealed that MTAN had much higher diversity than AdoHcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS(+) bacteria could be discriminated from LuxS(-) bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle.

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus.

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor's C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.

Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation.

Occurrence of psychrotolerant Bacillus cereus group strains in ice creams.

The occurrences of Bacillus cereus group strains in 40 ice cream samples were investigated. Among 109 isolated B. cereus group strains confirmed by 16S rDNA sequence analysis only 50 were identified as B. cereus and one as B. thuringiensis by using FDA (U.S. Food and Drug Administration) standard, indicating the two identification standards were highly inconsistent. Furthermore, the psychrotolerant growth properties and the occurrence of specific psychrotolerant genes of the isolates were also studied. Both psychrotolerant 16S rDNA fragments and enterotoxic genes could be detected among mesophilic and psychrotolerant strains. No relationship among psychrotolerance, presence of psychrotolerant 16S rDNA fragments and enterotoxic genes were found and the specific cspA fragment was only detected in a small fraction (9.5%) of the psychrotolerant isolates. One psychrotolerant isolate Bw2-1 was identified as B. weihenstephanensis, but no clear distinguishing characteristics between B. weihenstephanensis and psychrotolerant B. cereus were found. These results might be of importance for gaining further understanding of the growth properties of B. weihenstephanensis and psychrotolerant B. cereus as well as their contribution to food poisoning.

Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41.

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K(m) values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.

Co-expression of the mosquitocidal toxins Cyt1Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus.

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.