Endoscopic imaging modalities for diagnosing the invasion depth of superficial esophageal squamous cell carcinoma: a systematic review

Esophagus - Endoscopic diagnosis of the invasion depth of superficial esophageal squamous cell carcinoma (ESCC) is an important determinant of the treatment strategy. The three endoscopic imaging...     

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.     

Antagonistic regulation of myogenesis by two deubiquitinating enzymes,UBP45 and UBP69

Protein modification by ubiquitin is a dynamic and reversible process that is involved in the regulation of a variety of cellular processes. Here, we show that myogenic differentiation of embryonic muscle cells is antagonistically regulated by two deubiquitinating enzymes, UBP45 and UBP69, that are generated by alternative splicing. Both enzymes cleaved off ubiquitin from polyubiquitinated protein conjugates in vivo as well as from linear ubiquitin-protein fusions in vitro. In cultured myoblasts, the level of UBP69 mRNA markedly but transiently increased before membrane fusion, whereas that of UBP45 mRNA increased as the cells fused to form myotubes. Both myoblast fusion and accumulation of myosin heavy chain were dramatically stimulated by the stable expression of UBP69 but strongly attenuated by that of the catalytically inactive form of the protease, suggesting that the mutant enzyme acts dominant negatively on the function of the wild-type protease. In contrast, stable expression of UBP45 completely blocked both of the myogenic processes but that of inactive enzyme did not, indicating that the catalytic activity of the enzyme is essential for its inhibitory effects. These results indicate that differential expression of UBP45 and UBP69 is involved in the regulation of muscle cell differentiation.     

High seroprevalence of anti–HTLV-I antibody in rheumatoid arthritis

Objective. To investigate the association between human T lymphotropic virus type I (HTLV-I) infection and rheumatoid arthritis (RA) in Nagasaki, an area highly endemic for HTLV-I infection. Methods. Sera from 113 female patients with RA and 19,796 female blood donors were screened for anti–HTLV-I antibodies with a gelatin particle agglutination kit and confirmed using an immunoblotting kits. Results. The age-adjusted summary odds ratio of HTLV-I infection among RA patients, as compared with blood donors, was 2.8 (95% confidence interval [95% CI] 1.8–4.6). The etiologic fraction, i.e., the proportion of RA in the study population that is attributable to HTLV-I infection, was estimated to be 13.2% (95% CI 5.1–21.2). There was no significant difference in the clinical and laboratory findings between HTLV-I–infected and HTLV-I–uninfected RA patients. Conclusion. These epidemiologic findings support the idea that HTLV-I infection is a risk factor for RA, and suggest that ∼13% of the cases of RA in females living in Nagasaki are associated with HTLV-I infection.