The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a <40-fold increase in the 50% lethal dose by intravenous inoculation. Therefore, CRP is required for the virulence of Y. pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

Biopolymer-based supramolecular hydrogels cross-linked by host−guest interactions are usually mechanically weak as shown in "inverted vials" instead of freestanding 3D constructs. Herein, we describe a novel host−guest macromer (HGM) approach for preparation of biopolymerbased freestanding supramolecular hydrogels. Host−guest macromers are formed by molecular self-assembly between adamantane-functionalized hyaluronic acid (AD x HA) guest polymers and monoacrylated β-cyclodextrins (mono-Ac-βCD) host monomers. Supramolecular hydrogels are readily prepared by UV-induced polymerization of the preassembled host−guest macromers. Such hydrogels are soely cross-linked by in situ formed multivalent host−guest nanoclusters and show significantly reinforced mechanical properties yet still retain desirable supramolecular features. They can self-heal and be remolded into freestanding 3D constructs which afford effective protection on the encapsulated stem cells during the compression remolding, making them promising carriers for therapeutic cells that can quickly adapt to and integrate with surrounding tissues of the targeted defects. We demonstrate that such hydrogels not only sustain extended release of encapsulated proteinaceous growth factors (TGF-β1) but also support chondrogenesis of the human mesenchymal stem cells (hMSCs) and promote cartilage regeneration in a rat model.

SummaryRapid industrialization and urbanization during recent decades are having dramatic effects on urban soil properties and lead to large discharges of pollutants, which inevitably affect the health of the soil, ecosystems and human populations. This paper provides a systematic review of the relations between urban soil and human health. First, it summarizes the organic and inorganic pollutants in urban soil and their potential risks to human health. Second, the relations between urban greenbelt land, soil microbial diversity and human health are also explored. Third, we propose that future research should focus on the integration of assessments of health risks with exposure pathways and site characteristics. Bioavailability-based risk assessment frameworks for pollutants in urban soil can elucidate the complicated relations between urban soil, pollutant exposure and human health in cities. Finally, management of urban soil and policy should be strengthened in the future to maintain its sustainable development and utilization. More effort should be directed to understanding the relations between soil microbial diversity, green space and human health in cities.

Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L−1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L−1) and daidzin (73.2 mg L−1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.

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By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.

Since phenotype-based screening directly evaluates capability of small molecules for modulating biology in actual biological systems, it has become an important discover modality in modern pharmaceutical sciences. However, in order to fully elucidate the molecular mechanism underlying the bioactivity of small molecules, identification of their biological targets is an indispensable step. Among the many target identification strategies developed during the past several decades, affinity purification remains to be one of the most important and powerful approaches, as it can directly reveal the physical interactions between small molecules and their biomolecular targets. However, due to the complexity of the proteome and the diversity of small molecule-protein interactions, affinity purification faces the specificity challenge: how to identify the true specific targets from the non-specific background? Focusing on this challenge, in this review, we briefly introduce the history and background of affinity purification, and then we discussed the major technological developments aiming to address this challenge. We have summarized these approaches in two categories: noise reduction and comparative distinction. This review also highlights the importance of choosing an integrated approach combining multiple methods to achieving success in target identification.

Morphine can promote the pathogenesis of human acquired immunodeficiency syndrome through binding to the mu opioid receptor (MOR) in immune cells. Previous investigation has suggested that expression of the MOR gene in lymphocytes is triggered by cooperative interaction between transcription factors, specificity protein 1 (Sp1) and Ying Yang 1 (YY1), in the promoter region. However, the specific molecular mechanism by which immunodeficiency virus infection impacts regulation of the MOR gene expression in lymphocytes is still unclear. In this study, it was demonstrated that SIV (SIVmac239) infection may result in gradual reduction of the MOR gene expression and Sp1 during a period of 48 h postinfection by analysis of quantitative real-time RT-PCR and Western blotting. The results of methylation-specific PCR showed that two of 14 CpG islands adjacent to the Sp1 and YY1 elements in the promoter region were methylated, which together with reduced Sp1, contributed to the failure of interaction of Sp1 with YY1 and their binding to the elements, as determined by coimmunoprecipitation, chromatin immunoprecipitation-real-time PCR, and EMSAs. The repression of the MOR gene secondary to SIVmac239 infection could be abolished by the demethylating agent 5-aza-2'-deoxycytidine. Transfection with Sp1-expressing vector (PN3-Sp1) was also able to enhance the activity of the promoter in SIVmac239-infected cells. We therefore concluded that aberrant methylation of the promoter and reduction of Sp1 resulting from SIVmac239 infection led to the silencing of the MOR gene. This finding will be helpful in understanding the synergistic mechanism of HIV infection and morphine addiction in the pathogenesis of AIDS.