Molecularly Imprinted Macroporous Hydrogel Promotes Bone Regeneration via Osteogenic Induction and Osteoclastic Inhibition.

Macroporous hydrogels offer physical supportive spaces and bio-instructive environment for the seeded cells, where cell-scaffold interactions directly influence cell fates and subsequently affect tissue regeneration post-implantation. Effectively modifying bioactive motifs at the inner pore surface provides appropriate niches for cell-scaffold interactions. A molecular imprinting method and sacrificial templates are introduced to prepare inner pore surface modification in the macroporous hydrogels. In detail, acrylated bisphosphonates (Ac-BPs) chelating to templates (CaCO3 particles) are anchored on the inner pore surface of the methacrylated gelatin (GelMA)-methacrylated hyaluronic acid (HAMA)-poly (ethylene glycol) diacrylate (PEGDA) macroporous hydrogel (GHP) to form a functional hydrogel scaffold (GHP-int-BP). GHP-int-BP, but not GHP, effectively crafts artificial cell niches to substantially alter cell fates, including osteogenic induction and osteoclastic inhibition, and promote in situ bone regeneration. These findings highlight that molecular imprinting on the inner pore surface in the hydrogel efficiently creates orthogonally additive bio-instructive scaffolds for bone regeneration.

Double heterozygous pathogenic mutations in KIF3C and ZNF513 cause hereditary gingival fibromatosis.

Hereditary gingival fibromatosis (HGF) is a rare inherited condition with fibromatoid hyperplasia of the gingival tissue that exhibits great genetic heterogeneity. Five distinct loci related to non-syndromic HGF have been identified; however, only two disease-causing genes, SOS1 and REST, inducing HGF have been identified at two loci, GINGF1 and GINGF5, respectively. Here, based on a family pedigree with 26 members, including nine patients with HGF, we identified double heterozygous pathogenic mutations in the ZNF513 (c.C748T, p.R250W) and KIF3C (c.G1229A, p.R410H) genes within the GINGF3 locus related to HGF. Functional studies demonstrated that the ZNF513 p.R250W and KIF3C p.R410H variants significantly increased the expression of ZNF513 and KIF3C in vitro and in vivo. ZNF513, a transcription factor, binds to KIF3C exon 1 and participates in the positive regulation of KIF3C expression in gingival fibroblasts. Furthermore, a knock-in mouse model confirmed that heterozygous or homozygous mutations within Zfp513 (p.R250W) or Kif3c (p.R412H) alone do not led to clear phenotypes with gingival fibromatosis, whereas the double mutations led to gingival hyperplasia phenotypes. In addition, we found that ZNF513 binds to the SOS1 promoter and plays an important positive role in regulating the expression of SOS1. Moreover, the KIF3C p.R410H mutation could activate the PI3K and KCNQ1 potassium channels. ZNF513 combined with KIF3C regulates gingival fibroblast proliferation, migration, and fibrosis response via the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways. In summary, these results demonstrate ZNF513 + KIF3C as an important genetic combination in HGF manifestation and suggest that ZNF513 mutation may be a major risk factor for HGF.

Comprehensive analysis of the long noncoding RNA-associated competitive endogenous RNA network in the osteogenic differentiation of periodontal ligament stem cells.

BACKGROUD: The mechanism implicated in the osteogenesis of human periodontal ligament stem cells (PDLSCs) has been investigated for years. Previous genomics data analyses showed that long noncoding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) have significant expression differences between induced and control human PDLSCs. Competing for endogenous RNAs (ceRNA), as a widely studied mechanism in regenerative medicine, while rarely reported in periodontal regeneration. The key lncRNAs and their ceRNA network might provide new insights into molecular therapies of periodontal regeneration based on PDLSCs. RESULTS: Two networks reflecting the relationships among differentially expressed RNAs were constructed. One ceRNA network was composed of 6 upregulated lncRNAs, 280 upregulated mRNAs, and 18 downregulated miRNAs. The other network contained 33 downregulated lncRNAs, 73 downregulated mRNAs, and 5 upregulated miRNAs. Functional analysis revealed that 38 GO terms and 8 pathways related with osteogenesis were enriched. Twenty-four osteogenesis-related gene-centred lncRNA-associated ceRNA networks were successfully constructed. Among these pathways, we highlighted MAPK and TGF-beta pathways that are closely related to osteogenesis. Subsequently, subnetworks potentially linking the GO:0001649 (osteoblast differentiation), MAPK and TGF-beta pathways were constructed. The qRT-PCR validation results were consistent with the microarray analysis. CONCLUSION: We construct a comprehensively identified lncRNA-associated ceRNA network might be involved in the osteogenesis of PDLSCs, which could provide insights into the regulatory mechanisms and treatment targets of periodontal regeneration.

Discovery and Validation of an Epithelial-Mesenchymal Transition-Based Signature in Gastric Cancer by Genomics and Prognosis Analysis.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) exerts a key function in cancer initiation and progression. Herein, we aimed to develop an EMT-based prognostic signature in gastric cancer. METHODS: The gene expression profiles of gastric cancer were obtained from TCGA dataset as a training set and GSE66229 and GSE84437 datasets as validation sets. By LASSO regression and Cox regression analyses, key prognostic EMT-related genes were screened for developing a risk score (RS) model. Potential small molecular compounds were predicted by the CMap database based on the RS model. GSEA was employed to explore signaling pathways associated with the RS. ESTIMATE and seven algorithms (TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL, and EPIC) were applied to assess the RS and immune microenvironment. RESULTS: This study developed an EMT-related gene signature comprised of SERPINE1, PCOLCE2, MATN3, and DKK1. High-RS patients displayed poorer survival outcomes than those with low RS. ROC curves demonstrated the robustness of the model in predicting the prognosis. After external validation, the RS model was an independent risk factor for gastric cancer. Several compounds were predicted for gastric cancer treatment based on the RS model. ECM receptor interaction, focal adhesion, pathway in cancer, TGF-beta, and WNT pathways were distinctly activated in high-RS samples. Also, high RS was significantly associated with increased stromal and immune scores and increased infiltration of CD4+ T cell, CD8+ T cell, cancer-associated fibroblast, and macrophage in gastric cancer tissues. CONCLUSION: Our findings suggested that the EMT-related gene model may robustly predict gastric cancer prognosis, which could improve the efficacy of personalized therapy.

Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus.

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

Long non-coding RNA MEG3 inhibits neovascularization in diabetic retinopathy by regulating microRNA miR-6720-5p and cytochrome B5 reductase 2.

Diabetic retinopathy (DR) is a major cause of vision loss in working and elderly populations. long non-coding RNA (LncRNA) MEG3 is thought to have some effect on DR, but the exact mechanism remains to be clarified. The expression levels of lncRNA MEG3, miR-6720-5p, and cytochrome B5 reductase 2 (CYB5R2) in human retinal microvascular endothelial cells (hRMECs) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), transwell migration, and tube formation assays were used to determine the cell viability, migration, and tube formation ability of hRMECs, respectively. The interaction of MEG3, miR-6720-5p, and CYB5R2 was detected and explored by a luciferase assay. The expression of MEG3 and CYB5R2 was upregulated and that of miR-6720-5p was downregulated in patients with DR and hRMECs treated with high glucose. Knocking down MEG3 or CYB5R2 promoted proliferation, migration, and neovascularization in hRMECs. The intervention of miR-6720-5p reversed the effect of MEG3 knockdown on hRMEC function, and this effect was eliminated by silencing CYB5R2. Therefore, MEG3 acted as a sponge to suppress miR-6720-5p and regulate the expression of CYB5R2, thereby inhibiting DR neovascularization.

Characterization of Lysobacter spp. strains and their potential use as biocontrol agents against pear anthracnose.

Colletotrichum fructicola, is an important fungal pathogen that has been reported to cause pear (Pyrus) anthracnose in China, resulting in substantial economic losses due to severe defoliation and decreased fruit quality and yield. In the search for novel strategies to control pear anthracnose, Lysobacter strains have drawn a great deal of attention due to their high-level production of extracellular enzymes and bioactive metabolites. In the present study, we compared four Lysobacter strains including Lysobacter enzymogenes OH11, Lysobacter antibioticus OH13, Lysobacter gummosus OH17 and Lysobacter brunescens OH23 with respect to their characteristics and activity against pear anthracnose caused by C. fructicola. The results showed that the evaluated Lysobacter species presented various colony morphologies when cultured on different media and were proficient in producing protease, chitinase, cellulase and glucanase, with L. enzymogenes OH11 showing typical twitching motility. L. enzymogenes OH11 and L. gummosus OH17 showed potent activity against the tested fungi and oomycetes. L. gummosus OH17 produced HSAF (heat-stable antifungal factor) which was demonstrated to be a major antifungal factor in L. enzymogenes OH11 and C3. Furthermore, L. antibioticus OH13 and L. brunescens OH23 exhibited strong antibacterial activity, especially against Xanthomonas species. Cultures of L. enzymogenes OH11 protected pear against anthracnose caused by C. fructicola, and the in vivo results indicated that treatment with an L. enzymogenes OH11 culture could decrease the diameter of lesions in pears by 35 % and reduce the severity of rot symptoms compared to that observed in the control. In the present study, we systemically compared four Lysobacter strains and demonstrated that they have strong antagonistic activity against a range of pathogens, demonstrating their promise in the development of biological control agents.

Resolvin D2 prevents inflammation and oxidative stress in the retina of streptozocin-induced diabetic mice.

Diabetic retinopathy is the main ocular complication of diabetes mellitus. The aim of this study was to investigate the protective effect and mechanism of resolvin D2 (RvD2) on diabetic retinopathy. Streptozocin-induced C57/BJ diabetic mice were divided into three groups: normal control, diabetes mellitus, and diabetes plus RvD2 treatment. After three months of diabetic model induction, exogenous RvD2 was injected, monthly for three months, into the vitreous cavity of mice in the diabetic treatment group. Retinal vascular leakage, ganglion cell apoptosis, inflammatory factor expression, and oxidative stress factors were detected one month after the last injection. The levels of retinal vascular leakage and ganglion cell apoptosis in diabetic mice treated with RvD2 were significantly lower than those in untreated diabetic mice, as were the retinal levels of inflammatory factors and oxidative stress. In conclusion, RvD2 might be used as a retinal protective factor for diabetes mellitus by reducing inflammation and oxidative stress.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Characterization of Odontogenic Differentiation from Human Dental Pulp Stem Cells Using TMT-Based Proteomic Analysis.

BACKGROUND: The repair of dental pulp injury relies on the odontogenic differentiation of dental pulp stem cells (DPSCs). To better understand the odontogenic differentiation of DPSCs and identify proteins involved in this process, tandem mass tags (TMTs) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to compare the proteomic profiles of induced and control DPSCs. METHODS: The proteins expressed during osteogenic differentiation of human DPSCs were profiled using the TMT method combined with LC-MS/MS analysis. The identified proteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Then, a protein-protein interaction (PPI) network was constructed. Two selected proteins were confirmed by western blotting (WB) analysis. RESULTS: A total of 223 proteins that were differentially expressed were identified. Among them, 152 proteins were significantly upregulated and 71 were downregulated in the odontogenic differentiation group compared with the control group. On the basis of biological processes in GO, the identified proteins were mainly involved in cellular processes, metabolic processes, and biological regulation, which are connected with the signaling pathways highlighted by KEGG pathway analysis. PPI networks showed that most of the differentially expressed proteins were implicated in physical or functional interaction. The protein expression levels of FBN1 and TGF-ß2 validated by WB were consistent with the proteomic analysis. CONCLUSIONS: This is the first proteomic analysis of human DPSC odontogenesis using a TMT method. We identified many new differentially expressed proteins that are potential targets for pulp-dentin complex regeneration and repair.

Control of Wheat Fusarium Head Blight by Heat-Stable Antifungal Factor (HSAF) from Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which belongs to the polycyclic tetramate macrolactam family, was isolated from Lysobacter enzymogenes fermentations and exhibited inhibitory activities against a wide range of fungal pathogens. In this study, the antifungal activity of HSAF against Fusarium graminearum in vitro and in vivo was investigated. A total of 50% of mycelial growth of F. graminearum was suppressed with 4.1 µg/ml of HSAF (EC50 value). HSAF treatment resulted in abnormal morphology of the hyphae, such as curling, apical swelling, and depolarized growth. Furthermore, HSAF adequately inhibited conidial germination and conidiation of F. graminearum with an inhibition rate of 100% when 1 and 6 µg/ml of HSAF were applied, respectively. HSAF caused ultrastructural changes of F. graminearum, including cell wall thickening and plasmolysis. Moreover, the application of HSAF significantly controlled Fusarium head blight in wheat caused by F. graminearum in the field. Overall, these results indicate that HSAF has potential for development as a fungicide against F. graminearum.

Toxicity of a molluscicide candidate PPU07 against Oncomelania hupensis (Gredler, 1881) and local fish in field evaluation.

Schistosomiasis japonica caused by Schistosoma japonicum infection is recognized as a considerable economic and public health concern in Asia. Oncomelania hupensis is the sole intermediate host of S. japonicum. The only molluscicide recommended by World Health Organization (WHO) since 1960s is relative toxic to other aquatic species. In this article, we evaluated the novel molluscicide PPU07 in field trials on their efficiency against O. hupensis and toxicity for local fish. 25% PPU07 sulfate WP exhibited similar molluscicidal effect at 2.0 g/m2 and 2.0 g/m3 in the spraying and immersion trials with the WHO recommended molluscicide niclosamide (1 g/m2 and 1 g/m3). The mortality rates reached 95% and 96%, respectively. Moreover, little toxicity was observed for local fish and other aquatic organisms at the effective molluscicidal concentrations. In all, 25% PPU07 sulfate WP is a promising molluscicide for snail control, particularly in semi-commercial or commercial aquaculture ponds.

Efficient production of heat-stable antifungal factor through integrating statistical optimization with a two-stage temperature control strategy in Lysobacter enzymogenes OH11.

BACKGROUND: Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production. RESULTS: Medium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26 ± 16.14 mg L- 1, increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%). CONCLUSIONS: This study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF.

Mussel-inspired dual-functional PEG hydrogel inducing mineralization and inhibiting infection in maxillary bone reconstruction.

Infection compromises healing process after bone fracture. An anti-bacterial bone graft synthesized from polymer and mineralization components is becoming preferable for its accessibility and low cost and tunable chem-physic properties. In this study, mussel-inspired polydopamine (PDA) was used to synthesize in-situ silver nanoparticles (AgNPs) and mineralization on polyethylene hydrogel (PEG). With dual functions of anti-bacteria and graft mineralization, we found the hydrogel (AgNPs/PDA) promoted bone generation and show significant antibacterial activity. Specifically, the gel upregulated the expression of osteogenic genes of bone sialoprotein gene, alkaline phosphatase, osteocalcin and runt-related transcription factor 2. It also significantly inhibited the growth of Staphylococcus aureus and Escherichia coli. In vivo the AgNPs/PDA gel could repair maxillary bone defect efficiently.

LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11.

BACKGROUND: Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp. RESULTS: Here, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level. CONCLUSIONS: These results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.

Lsp family proteins regulate antibiotic biosynthesis in Lysobacter enzymogenes OH11.

Ax21 family proteins have been shown to play regulatory roles in plant- and animal-pathogenic species in the bacterial family Xanthomonadaceae, but the protein have not been investigated previously in the non-pathogenic members of this bacterial family. Lysobacter enzymogenes, is a non-pathogenic species known for its capacity as a biocontrol agent of plant pathogens. It is also noted for the production of antimicrobial secondary metabolites, heat stable antifungal factor (HSAF) and WAP-8294A2, that have potential for agricultural and pharmaceutical applications. The species also displays type IV pili-dependent twitching motility and the production of multiple extracellular lytic enzymes as additional biocontrol-related traits. Here, we show that L. enzymogenes strain OH11 possesses three genes widely separated in the OH11 genome that code for unique Ax21-like proteins (Lsp). By comparing the wildtype OH11 with mutant strains having a single lsp gene or a combination of lsp genes deleted, we found that each Lsp protein individually is involved in positive regulation of HSAF and WAP-8294A2 biosynthesis, but the proteins collectively do not exert additive effects in this regulation. None of the Lsp proteins were found to influence twitching motility or the production of three extracellular lytic enzymes. This study is the first to provide evidence linking Ax21-family proteins to antibiotic biosynthesis and, hence, adds new insights into the diversity of regulatory functions of Ax21 family proteins in bacteria.

Periostin is essential for periodontal ligament remodeling during orthodontic treatment.

Orthodontic tooth movement is a process stimulated and maintained by external tensile stress; periodontal ligament remodeling serves an important role during this process. However, the function and underlying mechanism of periostin (PN) during orthodontic periodontal ligament remodeling remain unclear. The present study established in vitro and in vivo models of orthodontic treatment to investigate the expression levels of PN under conditions of external tensile stress load. These results indicated that tensile stress load increased the expression levels of PN in mouse peridontal ligaments and human periodontal ligament cells (hPDLCs), during orthodontic tooth movement. Furthermore, the present study demonstrated that the expression levels of PN were regulated by transforming grown factor ß, and that PN promotes type I collagen and α­smooth muscle actin expression levels in hPDLCs. Therefore, PN may be essential for periodontal ligament remodeling during orthodontic treatment, and therefore may represent a potential therapeutic target.

Direct Regulation of Extracellular Chitinase Production by the Transcription Factor LeClp in Lysobacter enzymogenes OH11.

Lysobacter enzymogenes is a gram-negative bacterial biological control agent that produces abundant extracellular enzymes capable of degrading the cell walls of fungal pathogens. In strain OH11, an isolate from China, the global regulator LeClp controls the production of extracellular chitinase by regulating the transcription of the chitinase-encoding gene chiA. Using a combination of bioinformatic, genetic, and biochemical methods, we show that LeClp regulates chiA transcription by directly binding to the chiA promoter region. Although LeClp appears to be important in this role, it is not the sole regulator of chiA transcription. Furthermore, the sequence analysis of putative LeClp binding sites indicated that the LeClp homolog could be involved in the regulation of extracellular chitinase production in diverse Lysobacter spp. by a mechanism similar to that in L. enzymogenes. Our findings present new insights into the molecular mechanism of LeClp in controlling extracellular chitinase activity, providing a fundamental road to elucidate how LeClp regulates the production of other extracellular lytic enzymes in L. enzymogenes.

A TonB-dependent receptor regulates antifungal HSAF biosynthesis in Lysobacter.

Lysobacter species are Gram-negative bacteria that are emerging as new sources of antibiotics, including HSAF (Heat Stable Antifungal Factor), which was identified from L. enzymogenes with a new mode of action. LesR, a LuxR solo, was recently shown to regulate the HSAF biosynthesis via an unidentified mechanism in L. enzymogenes OH11. Here, we used a comparative proteomic approach to identify the LesR targets and found that LesR influenced the expression of 33 proteins belonging to 10 functional groups, with 9 proteins belonging to the TBDR (TonB-Dependent Receptor) family. The fundamental role of bacterial TBDR in nutrient uptake motivates us to explore their potential regulation on HSAF biosynthesis which is also modulated by nutrient condition. Six out of 9 TBDR coding genes were individually in-frame deleted. Phenotypic and gene-expression assays showed that TBDR7, whose level was lower in a strain overexpressing lesR, was involved in regulating HSAF yield. TBDR7 was not involved in the growth, but played a vital role in transcribing the key HSAF biosynthetic gene. Taken together, the current lesR-based proteomic study provides the first report that TBDR7 plays a key role in regulating antibiotic (HSAF) biosynthesis, a function which has never been found for TBDRs in bacteria.

XocR, a LuxR solo required for virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, a serious bacterial disease of rice in Asia and parts of Africa. The virulence mechanisms of Xoc are not entirely clear and control measures for BLS are poorly developed. The solo LuxR proteins are widespread and shown to be involved in virulence in some plant associated bacteria (PAB). Here, we have cloned and characterized a PAB LuxR solo from Xoc, named as XocR. Mutation of xocR almost completely impaired the virulence ability of Xoc on host rice, but did not alter the ability to trigger HR (hypersensitive response, a programmed cell death) on non-host (plant) tobacco, suggesting the diversity of function of xocR in host and non-host plants. We also provide evidence to show that xocR is involved in the regulation of growth-independent cell motility in response to a yet-to-be-identified rice signal, as mutation of xocR impaired cell swimming motility of wild-type Rs105 in the presence but not absence of rice macerate. We further found that xocR regulated the transcription of two characterized virulence-associated genes (recN and trpE) in the presence of rice macerate. The promoter regions of recN and trpE possessed a potential binding motif (an imperfect pip box-like element) of XocR, raising the possibility that XocR might directly bind the promoter regions of these two genes to regulate their transcriptional activity. Our studies add a new member of PAB LuxR solos and also provide new insights into the role of PAB LuxR solo in the virulence of Xanthomonas species.