The utility of exhaled breath condensate analysis for asthma management

Asthma is a chronic inflammatory disorder of the airways; therefore, establishing a simple monitoring system of airway inflammation would be useful for asthma management. Exhaled breath condensate (EBC), which is formed by breathing with a cooling system, is a non-invasive biomarker for airway inflammation. We have shown that nine kinds of molecules; namely, IL-4, IL-8, IL-17, TNF-alpha, RANTES, IP-10, TGF-beta, MIP-1alpha, and MIP-1beta, are over-expressed in asthmatic airways compared with those of healthy subjects using EBC analysis. In addition, the levels of some over-expressed molecules and lung physiologic indices are correlated. RANTES expression was significantly correlated with the degree of airflow limitation. Further, both TNF-alpha and TGF-beta values were significantly correlated with the degree of airway responsiveness and variability of peak expiratory flow. Additionally, the comparison of molecular properties between EBC and saliva indicated that saliva contamination is not a dominant contributor to the assessment of inflammatory molecules in EBC. Analysis of inflammatory molecules using EBC should be useful for monitoring the asthmatic airway condition, and might be a promising approach to assess the efficacy of pharmacologic interventions and to investigate the pathophysiology of asthma.     

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D–treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Osteoporosis is a common cause of bone fracture in the elderly, costing billions globally due to fractures leading to long-term disability and subsequent exit from the working population (1, 2). Several treatment options are available for osteoporosis which can be divided into antiresorptives and anabolics ranging from orally dosed small molecules to injectable peptides and biologics (2, 3). Antiresorptive and anabolic agents differ in that antiresorptives inhibit or reduce bone resorption, thereby suppressing bone remodeling, whereas anabolics enhance the rate of bone formation while allowing continued resorption and remodeling of bone tissue. Although both treatments result in increased bone mass, resorption and remodeling are key to the microstructural integrity of bone, and emerging evidence points toward anabolics being more effective in reducing fracture (4–6). Currently, the only anabolics in the clinic are the parathyroid hormone and parathyroid hormone-related peptide mimetics (teriparatide and abaloparatide, respectively) and the sclerostin inhibitor monoclonal antibody (romosozumab). Teriparatide and abaloparatide both cannot be administered over 24 mo in a patient’s lifetime due to the risk for developing osteosarcomas, and romosozumab treatment is recommended for 12 mo due to waning efficacy beyond this duration (7, 8). Therefore, with the ever increasing global median age and associated osteoporosis cases, the development of additional anabolic treatment options are of high importance.Bone resorption and bone formation are regulated through communications between osteoclasts and osteoblasts, respectively (9). Among the paracrine factors involved in this process, axon guidance molecules, such as Semaphorin4D (Sema4D) and Semaphorin3A, mediate the regulation of bone cell differentiation. Sema4D, which is expressed and secreted by mature osteoclasts, inhibits osteoblast differentiation through its receptor PlexinB1 (PlxnB1) expressed on osteoblast surfaces. Binding of Sema4D to PlxnB1 leads to the inhibition of the activation of insulin receptor substrate-1 which is downstream of insulin-like growth factor-1 signaling. In addition, Sema4D controls the spatial distribution of bone-forming osteoblasts through PlxnB1-RhoA signaling (10). Mice with genetic deletion of Sema4D or PlxnB1 as well as mice expressing a dominant-negative form of RhoA in osteoblasts exhibit a high bone mass phenotype due to increased bone formation (11). These findings suggest that inhibiting PlxnB1-Sema4D signaling would lead to a bone anabolic effect through enhancement of osteoblastic differentiation while keeping osteoblasts away from osteoclasts to enable efficient osteoclastic bone resorption.We have previously reported a macrocyclic peptide discovery campaign to identify binding sites on PlxnB1 that inhibit its interaction with Sema4D by means of messenger RNA (mRNA) display in combination with genetic code reprogramming, referred to as the RaPID (Random nonstandard Peptides Integrated Discovery) system (12). We successfully identified a 16-mer thioether-macrocyclic peptide, PB1m6, capable of binding human PlxnB1 (hPlxnB1) with single-digit nanomolar-binding affinity and inhibiting its interaction with Sema4D (13). X-ray structural analysis of cocrystals of PB1m6 and hPlxnB1 revealed that PB1m6 is a negative allosteric modulator of the hPlxnB1-Sema4D interaction by binding a cleft distal to the Sema4D-binding interface of hPlxnB1 while still able to inhibit the hPlxnB1-Sema4D interaction. However, PB1m6 was shown to be selective toward hPlxnB1 over mouse PlxnB1 (mPlxnB1) displaying no detectable affinity (dissociation constant (K_D) over 1 µM) regardless of having 88% sequence identity in the N-terminal sema domains of hPlxnB1 and mPlxnB1. Modeling efforts based on the three-dimensional (3D) structure to rationally increase the species cross-reactivity were unsuccessful in our hands. This high selectivity is often observed with RaPID-derived macrocyclic peptides and is generally considered beneficial (13–18). However, in this instance, the high selectivity of PB1m6 hinders its ability to validate the inhibitory mechanism in mouse models. In this study, we used a fragmented saturation mutagenesis approach to create an mRNA library of PB1m6 analogs to be utilized in a RaPID selection campaign against mouse PlxnB1. After five iterative rounds of selection, we discovered a PB1m6 analog, referred to as PB1m6A9, exhibiting enhanced cross-reactivity with 44 nM K_D against mouse PlxnB1 and which, remarkably, also showed 10-fold stronger binding affinity against human PlxnB1 (0.28 nM K_D). To further improve apparent affinity and inhibitory activity, a homodimer of PB1m6A9 was chemically synthesized (PB1d6A9), and it was shown to exhibit potent mPlxnB1-Sema4D inhibitory activity in mouse primary osteoblasts as well as enhanced osteogenesis even when compared to cells not treated with Sema4D. Moreover, once-weekly intravenous (i.v.) administration of palmitoylated PB1d6A9 (PB1d6A9-Pal) in a mouse model of postmenopausal osteoporosis showed significant enhancement of bone formation compared to both vehicle and sham-operated (Sham) control mice. This work presents the facile development of a novel bone anabolic modality which shows promise as an addition to the current repertoire of anabolic agents used to address osteoporosis.     

Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.

A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed.     

Comparative therapeutic effects of vitamin D3 and its derivatives on experimental renal osteodystrophy

The comparative effectiveness of vitamin D3 and its derivatives in curing hyperparathyroidism and osteodystrophic bone lesions was examined in a laboratory model of renal osteodystrophy associated with marked secondary hyperparathyroidism in rats. The experimental model was prepared by a single injection of homologous glycopeptide. Plasma levels of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] appeared to decrease in the rats receiving glycopeptide. Various doses of vitamin D3 derivatives [2 or 10 microgram/kg D3, 2 microgram/kg 25-hydroxyvitamin D3 (25OHD3), 0.1 microgram/kg 1 alpha,25(OH)2D3, and 0.1 or 0.2 microgram/kg 1 alpha-hydroxyvitamin D3 (1 alpha OHD3)] were daily administered orally to the nephritic rats for 23 days before sacrifice. 1 alpha,25(OH)2D3 and 1 alpha OHD3 were much more potent than 25OHD3 and D3 in reducing the hyperplasia of parathyroir glands. The potency of 1 alpha OHD3 in curing the histological changes of osteodystrophy appeared to be greater than that of 1 alpha,25(OH)2D3. The same dose level of 1 alpha OHD3 was more effective than 1 alpha,25(OH)2D3 in enhancing plasma 1 alpha,25(OH)2D3 levels.     

In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.     

Successful arthroscopic treatment of pigmented villonodular synovitis of the knee in a patient with congenital deficiency of plasminogen activator inhibitor-1 and recurrent haemarthrosis.

We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of "active PAI" in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.     

Cognitive behavioral therapy for depression changes medial prefrontal and ventral anterior cingulate cortex activity associated with self-referential processing

Cognitive behavioral therapy (CBT), an effective treatment for depression, targets self-referential processing of emotional stimuli. We examined the effects of CBT on brain functioning during self-referential processing in depressive patients using functional magnetic resonance imaging (fMRI). Depressive patients (n = 23) and healthy participants (n = 15) underwent fMRI scans during a self-referential task using emotional trait words. The depressive patients had fMRI scans before and after completing a total of 12 weekly sessions of group CBT for depression, whereas the healthy participants underwent fMRI scans 12 weeks apart with no intervention. Before undergoing CBT, the depressive patients showed hyperactivity in the medial prefrontal cortex (MPFC) during self-referential processing of negative words. Following CBT, MPFC and ventral anterior cingulate cortex (vACC) activity during self-referential processing among depressive patients was increased for positive stimuli, whereas it was decreased for negative stimuli. Improvements in depressive symptoms were negatively correlated with vACC activity during self-referential processing of negative stimuli. These results suggest that CBT-related improvements in depressive symptoms are associated with changes in MPFC and vACC activation during self-referential processing of emotional stimuli.     

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.With a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world''s most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography–mass spectrometry (HPLC-MS) system uses methods of extraction, chromatography, and databases that are specialized for mycobacteria. After extraction of total bacterial lipids into organic solvents, HPLC-MS enables massively parallel detection of thousands of ions corresponding to diverse lipids that range from apolar polyketides to polar phosphoglycolipids. Software-based (XCMS) ion finding algorithms report reproducibly detected ions as molecular features. Each feature is a 3D data point with linked mass, retention time, and intensity values from one detected molecule or isotope. All features with equivalent mass and retention time from two bacterial lipid extracts are aligned, allowing pairwise comparisons of MS signal intensity to enumerate molecules that are overproduced in one strain with a false-positive rate below 1% (4).This comparative lipidomics system allowed an unbiased, organism-wide analysis of lipids from M. tuberculosis and the attenuated vaccine strain, Mycobacterium bovis Bacillus Calmette–Guérin (BCG). BCG was chosen because of its worldwide use as a vaccine and its genetic similarity to M. tuberculosis (6). We reasoned that any features that are specifically detected in M. tuberculosis might be clinically useful as markers to distinguish tuberculosis-causing bacteria from vaccines. Furthermore, given the differing potential for productive infection by the two strains, any M. tuberculosis-specific compounds would be candidate virulence factors. Comparative genomics of M. tuberculosis and BCG successfully identified “regions of deletion” (RD) that encode genes that were subsequently proven to promote productive M. tuberculosis infection (7), including the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 (ESX-1) (8, 9). We reasoned that a metabolite-based screen might identify new virulence factors because not all functions of RD genes are known. Also, biologically important metabolites could emerge from complex biosynthetic pathways that cannot be predicted from single-gene analysis.Comparison of M. tuberculosis and BCG lipid profiles revealed more than 1,000 differences, among which we identified a previously unknown M. tuberculosis-specific diterpene-linked adenosine and showed that it is produced by the enzyme Rv3378c. Previously, Rv3378c was thought to generate free tuberculosinol and isotuberculosinol (10–12). This discovery revises the enzymatic function of Rv3378c, which acts as a virulence factor to inhibit phagolysosome fusion (13). Whereas current models of prenyl transferase function emphasize iterative lengthening of prenyl pyrophosphates using one binding pocket, the crystal structure of Rv3378c identifies two pockets in the catalytic site, establishing a mechanism for heterologous prenyl transfer to nonprenyl metabolites.