Osteoblast lineage cells and periodontal ligament fibroblasts regulate orthodontic tooth movement that is dependent on Nuclear Factor-kappa B (NF-kB) activation.

OBJECTIVES: To investigate the role of NF-κB in osteoblast lineage cells and periodontal ligament (PDL) fibroblasts during orthodontic tooth movement (OTM). MATERIALS AND METHODS: Transgenic mice that expressed a dominant negative mutant of the inhibitor of kB kinase (IKK-DN) with lineage specific expression in osteoblastic cells and PDL fibroblasts driven by a response element in the collagen1α1 promoter and matched wild-type (WT) mice were examined. A 10-12 g force was applied by a NiTi coil and maintained for 5 or 12 days. OTM distance, PDL width, and bone volume fraction were measured using micro computed tomography. Osteoclast numbers were counted in tartrate-resistant acid phosphatase-stained sections. Activation of nuclear factor kappa B (NF-kB) was assessed by nuclear localization of p65, and the receptor activator of nuclear factor-κB ligand (RANKL) was measured by immunofluorescence and compared to control specimens with no orthodontic force. RESULTS: OTM-induced NF-kB activation (p65 nuclear localization) in WT mice was largely blocked in transgenic (TG) mice. OTM was significantly reduced in the TG mice compared to WT mice along with reduced osteoclastogenesis, narrower PDL width, higher bone volume fraction, and reduced RANKL expression. CONCLUSIONS: Osteoblast lineage cells and PDL fibroblasts are key contributors to alveolar bone remodeling in OTM through IKKß dependent NF-κB activation.

Synthesis and analysis of 4-(3-fluoropropyl)-glutamic acid stereoisomers to determine the stereochemical purity of (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) for clinical use.

(4S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) is a positron emission tomography (PET) imaging agent for measuring the system xC- transporter activity. It has been used for the detection of various cancers and metastasis in clinical trials. [18F]FSPG is also a promising diagnostic tool for evaluation of multiple sclerosis, drug resistance in chemotherapy, inflammatory brain diseases, and infectious lesions. Due to the very short half-life (110 min) of 18F nuclide, [18F]FSPG needs to be produced on a daily basis; therefore, fast and efficient synthesis and analytical methods for quality control must be established to assure the quality and safety of [18F]FSPG for clinical use. To manufacture cGMP-compliant [18F]FSPG, all four nonradioactive stereoisomers of FSPG were prepared as reference standards for analysis. (2S,4S)-1 and (2R,4R)-1 were synthesized starting from protected L- and D-glutamate derivatives in three steps, whereas (2S,4R)-1 and (2R,4S)-1 were prepared in three steps from protected (S)- and (R)-pyroglutamates. A chiral HPLC method for simultaneous determination of four FSPG stereoisomers was developed by using a 3-cm Chirex 3126 column and a MeCN/CuSO4(aq) mobile phase. In this method, (2R,4S)-1, (2S,4S)-1, (2R,4R)-1, and (2S,4R)-1 were eluted in sequence with sufficient resolution in less than 25 min without derivatization. Scale-up synthesis of intermediates for the production of [18F]FSPG in high optical purity was achieved via stereo-selective synthesis or resolution by recrystallization. The enantiomeric excess of intermediates was determined by HPLC using a Chiralcel OD column and monitored at 220 nm. The nonradioactive precursor with >98% ee can be readily distributed to other facilities for the production of [18F]FSPG. Based on the above accomplishments, cGMP-compliant [18F]FSPG met the acceptance criteria in specifications and was successfully manufactured for human use. It has been routinely prepared and used in several pancreatic ductal adenocarcinoma metastasis-related clinical trials.

Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells.

Toll-like receptors (TLRs) are important sensors that recognize pathogen-associated molecular patterns. Generally, TLR9 is known to recognize bacterial or viral DNA but not viral RNA and initiate an immune response. Herein, we demonstrate that infection with dengue virus (DENV), an RNA virus, activates TLR9 in human dendritic cells (DCs). DENV infection induces release of mitochondrial DNA (mtDNA) into the cytosol and activates TLR9 signaling pathways, leading to production of interferons (IFNs). The DENV-induced mtDNA release involves reactive oxygen species generation and inflammasome activation. DENV infection disrupts the association between transcription factor A mitochondria (TFAM) and mtDNA and activates the mitochondrial permeability transition pores. The side-by-side comparison of TLR9 and cyclic GMP-AMP synthase (cGAS) knockdown reveals that both cGAS and TLR9 comparably contribute to DENV-induced immune activation. The significance of TLR9 in DENV-induced immune response is also confirmed in examination with the bone marrow-derived DCs prepared from Tlr9-knockout mice. Our study unravels a previously unrecognized phenomenon in which infection with an RNA virus, DENV, activates TLR9 signaling by inducing mtDNA release in human DCs.

RANKL deletion in periodontal ligament and bone lining cells blocks orthodontic tooth movement.

The bone remodeling process in response to orthodontic forces requires the activity of osteoclasts to allow teeth to move in the direction of the force applied. Receptor activator of nuclear factor-κB ligand (RANKL) is essential for this process although its cellular source in response to orthodontic forces has not been determined. Orthodontic tooth movement is considered to be an aseptic inflammatory process that is stimulated by leukocytes including T and B lymphocytes which are presumed to stimulate bone resorption. We determined whether periodontal ligament and bone lining cells were an essential source of RANKL by tamoxifen induced deletion of RANKL in which Cre recombinase was driven by a 3.2 kb reporter element of the Col1α1 gene in experimental mice (Col1α1.CreERTM+.RANKLf/f) and compared results with littermate controls (Col1α1.CreERTM-.RANKLf/f). By examination of Col1α1.CreERTM+.ROSA26 reporter mice we showed tissue specificity of tamoxifen induced Cre recombinase predominantly in the periodontal ligament and bone lining cells. Surprisingly we found that most of the orthodontic tooth movement and formation of osteoclasts was blocked in the experimental mice, which also had a reduced periodontal ligament space. Thus, we demonstrate for the first time that RANKL produced by periodontal ligament and bone lining cells provide the major driving force for tooth movement and osteoclastogenesis in response to orthodontic forces.

Identification of Buctopamine and Mebuctopamine, a ß2 Receptor Agonist and Its Metabolite, in Swine Hair and Feed Additives.

4-[2-(t-Butylamino)-1-hydroxyethyl]phenol (buctopamine, 4), a new ß2 receptor agonist (ß2-agonist), was found to be an adulterant in feed additives for swine in Taiwan, where using ß2-agonists in food-production animals is prohibited. Buctopamine and its metabolite, 4-[2-(t-butylamino)-1-hydroxyethyl]-2-methoxyphenol (mebuctopamine, 2), were detected in swine hair specimens. Authentic compounds 2 and 4 were synthesized with 98.6% and 97.7% purity, respectively, as reference standards for analysis, and both compounds were more hydrophilic than ractopamine and clenbuterol. In a preliminary pharmacological evaluation, compounds 2 and 4 exhibited moderate human ß2 receptor binding affinity and did not show significant affinities for the human α1, α2, ß1, and ß3 receptors. After addition of compounds 2-4 into the ß2-agonist library, a multiresidue analysis of 26 ß2-agonists by using triple quadrupole LC/MS/MS for routine screening conducted by regulatory authorities was established, in which the common limits of quantification for the 26 ß2-agonists in swine feed and hair are 10 and 25 ng/g, respectively. In addition, the illegal use of buctopamine (4) has been effectively prevented. The results of this study are also useful for controlling the illegal use of new ß2-agonists in food-production animals.

Fast and Facile Synthesis of 4-Nitrophenyl 2-Azidoethylcarbamate Derivatives from N-Fmoc-Protected α-Amino Acids as Activated Building Blocks for Urea Moiety-Containing Compound Library.

A fast and facile synthesis of a series of 4-nitrophenyl 2-azidoethylcarbamate derivatives as activated urea building blocks was developed. The N-Fmoc-protected 2-aminoethyl mesylates derived from various commercially available N-Fmoc-protected α-amino acids, including those having functionalized side chains with acid-labile protective groups, were directly transformed into 4-nitrophenyl 2-azidoethylcarbamate derivatives in 1 h via a one-pot two-step reaction. These urea building blocks were utilized for the preparation of a series of urea moiety-containing mitoxantrone-amino acid conjugates in 75-92% yields and parallel solution-phase synthesis of a urea compound library consisted of 30 members in 38-70% total yields.

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment.

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative 'omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available.

A theranostic micelleplex co-delivering SN-38 and VEGF siRNA for colorectal cancer therapy.

The development of an efficient colorectal cancer therapy is currently a public health priority. In the present work, we proposed a multifunctional theranostic micellar drug delivery system utilizing cationic PDMA-block-poly(ε-caprolactone) (PDMA-b-PCL) micelles as nanocarriers of SN-38 (7-ethyl-10-hydroxycamptothecin), ultra-small superparamagnetic iron oxide nanoparticles (USPIO), and small interfering RNA (siRNA) that targets human vascular endothelial growth factor (VEGF). The VEGF siRNA was conjugated to polyethylene glycol (PEG) (siRNA-PEG) before complexation with the micelles in order to improve the siRNA's stability and to prolong its retention time in the blood circulation. To further improve the in vivo biosafety, we prepared mixed micelles using mPEG-PCL together with PDMA-b-PCL copolymer. The SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes passively targeted to tumor regions and synergistically facilitated VEGF silencing and chemotherapy, thus efficiently suppressing tumor growth via a multi-dose therapy regimen. Additionally, the SN-38/USPIO-loaded siRNA-PEG mixed micelleplexes acted as a negative magnetic resonance imaging (MRI) contrast agent in T2-weighted imaging, resulting in a powerful tool for the diagnosis and for tracking of the therapeutic outcomes. In summary, we established a theranostic micellar drug and gene delivery system that not only synergistically combined gene silencing and chemotherapy but also served as a negative MRI contrast agent, which reveal its potential as a novel colorectal cancer therapy.

Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans.

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a ß-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care.

Mitochondria: signaling with phosphatidic acid.

Mitochondria, once viewed as functioning relatively autonomously in the cell, have increasingly been recognized to be involved in numerous signaling networks that impact on a wide range of cell biological processes. In addition to the many types of proteins that mediate these pathways, the importance of signaling functions regulated via lipids and lipid second messengers generated on the mitochondrial surface is also becoming well appreciated. We focus here on phosphatidic acid, a lipid second messenger produced via several different pathways that can in turn stimulate the formation of multiple other bioactive lipids. Taken together, fascinating roles for phosphatidic acid and the connected lipids in mitochondrial function and interaction with other organelles are being uncovered. These pathways present new opportunities for the development of therapeutic approaches relevant to reproduction, metabolism, and neurodegenerative disease.

Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy.

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

POINeT: protein interactome with sub-network analysis and hub prioritization.

BACKGROUND: Protein-protein interactions (PPIs) are critical to every aspect of biological processes. Expansion of all PPIs from a set of given queries often results in a complex PPI network lacking spatiotemporal consideration. Moreover, the reliability of available PPI resources, which consist of low- and high-throughput data, for network construction remains a significant challenge. Even though a number of software tools are available to facilitate PPI network analysis, an integrated tool is crucial to alleviate the burden on querying across multiple web servers and software tools. RESULTS: We have constructed an integrated web service, POINeT, to simplify the process of PPI searching, analysis, and visualization. POINeT merges PPI and tissue-specific expression data from multiple resources. The tissue-specific PPIs and the numbers of research papers supporting the PPIs can be filtered with user-adjustable threshold values and are dynamically updated in the viewer. The network constructed in POINeT can be readily analyzed with, for example, the built-in centrality calculation module and an integrated network viewer. Nodes in global networks can also be ranked and filtered using various network analysis formulas, i.e., centralities. To prioritize the sub-network, we developed a ranking filtered method (S3) to uncover potential novel mediators in the midbody network. Several examples are provided to illustrate the functionality of POINeT. The network constructed from four schizophrenia risk markers suggests that EXOC4 might be a novel marker for this disease. Finally, a liver-specific PPI network has been filtered with adult and fetal liver expression profiles. CONCLUSION: The functionalities provided by POINeT are highly improved compared to previous version of POINT. POINeT enables the identification and ranking of potential novel genes involved in a sub-network. Combining with tissue-specific gene expression profiles, PPIs specific to selected tissues can be revealed. The straightforward interface of POINeT makes PPI search and analysis just a few clicks away. The modular design permits further functional enhancement without hampering the simplicity. POINeT is available at (http://poinet.bioinformatics.tw/).

PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database.

MOTIVATION: To fully understand how a protein kinase regulates biological processes, it is imperative to first identify its substrate(s) and interacting protein(s). However, of the 518 known human serine/threonine/tyrosine kinases, 35% of these have known substrates, while 14% of the kinases have identified substrate recognition motifs. In contrast, 85% of the kinases have protein-protein interaction (PPI) datasets, raising the possibility that we might reveal potential kinase-substrate pairs from these PPIs. RESULTS: PhosphoPOINT, a comprehensive human kinase interactome and phospho-protein database, is a collection of 4195 phospho-proteins with a total of 15 738 phosphorylation sites. PhosphoPOINT annotates the interactions among kinases, with their down-stream substrates and with interacting (phospho)-proteins to modulate the kinase-substrate pairs. PhosphoPOINT implements various gene expression profiles and Gene Ontology cellular component information to evaluate each kinase and their interacting (phospho)-proteins/substrates. Integration of cSNPs that cause amino acids change with the proteins with the phosphoprotein dataset reveals that 64 phosphorylation sites result in a disease phenotypes when changed; the linked phenotypes include schizophrenia and hypertension. PhosphoPOINT also provides a search function for all phospho-peptides using about 300 known kinase/phosphatase substrate/binding motifs. Altogether, PhosphoPOINT provides robust annotation for kinases, their downstream substrates and their interaction (phospho)-proteins and this should accelerate the functional characterization of kinomemediated signaling. AVAILABILITY: PhosphoPOINT can be freely accessed in http://kinase. bioinformatics.tw/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation.

Production of a red blood cell's hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.