Synthesis of peptide conjugates with vitamins for induction of antigen‐specific immunotolerance

In this report, we designed conjugates of an antigen peptide with the immunosuppressive vitamins all‐trans retinoic acid (ATRA) and vitamin D3 for efficient induction of antigen‐specific immunotolerance. We established a synthetic scheme for the preparation of the peptide‐vitamin conjugates, which the chemically unstable vitamins tolerated. Among the obtained conjugates, the ATRA conjugate successfully suppressed inflammatory effects in macrophages and dendritic cells and induced antigen presentation in dendritic cells. This synthetic method of conjugate is conceivably applicable to other antigen peptides for induction of antigen‐specific immunotolerance.     

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 μM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.     

Spine and dine: A key defensive trait promotes ecological success in spiny ants

A key focus of ecologists is explaining the origin and maintenance of morphological diversity and its association with ecological success. We investigate potential benefits and costs of a common and varied morphological trait, cuticular spines, for foraging behavior, interspecific competition, and predator–prey interactions in naturally co‐occurring spiny ants (Hymenoptera: Formicidae: Polyrhachis) in an experimental setting. We expect that a defensive trait like spines might be associated with more conspicuous foraging, a greater number of workers sent out to forage, and potentially increased competitive ability. Alternatively, consistent with the ecological trade‐off hypothesis, we expect that investment in spines for antipredator defense might be negatively correlated with these other ecological traits. We find little evidence for any costs to ecological traits, instead finding that species with longer spines either outperform or do not differ from species with shorter spines for all tested metrics, including resource discovery rate and foraging effort as well as competitive ability and antipredator defense. Spines appear to confer broad antipredator benefits and serve as a form of defense with undetectable costs to key ecological abilities like resource foraging and competitive ability, providing an explanation for both the ecological success of the study genus and the large number of evolutionary origins of this trait across all ants. This study also provides a rare quantitative empirical test of ecological effects related to a morphological trait in ants.     

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

Focal articular cartilage damage can eventually lead to the onset of osteoarthritis with degradation around healthy articular cartilage. Currently, there are no drugs available that effectively repair articular cartilage damage. Several surgical techniques exist and are expected to prevent progression to osteoarthritis, but they do not offer a long‐term clinical solution. Recently, regenerative medicine approaches using human pluripotent stem cells (PSCs) have gained attention as new cell sources for therapeutic products. To translate PSCs to clinical application, appropriate cultures that produce large amounts of chondrocytes and hyaline cartilage are needed. So too are assays for the safety and efficacy of the cellular materials in preclinical studies including animal transplantation models. To confirm safety and efficacy, transplantation into the subcutaneous space and articular cartilage defects have been performed in animal models. All but one study we reviewed that transplanted PSC‐derived cellular products into articular cartilage defects found safe and effective recovery. However, for most of those studies, the quality of the PSCs was not verified, and the evaluations were done with small animals over short observation periods. Large animals and longer observation times are preferred. We will discuss the recent progress and future direction of the animal transplantation studies for the treatment of focal articular cartilage damages using PSCs.     

Mesorhizobium sp. J8 can establish symbiosis with Glycyrrhiza uralensis,increasing glycyrrhizin production

Licorice (Glycyrrhiza uralensis) is a medicinal plant that contains glycyrrhizin (GL), which has various pharmacological activities. Because licorice is a legume, it can establish a symbiotic relationship with nitrogen-fixing rhizobial bacteria. However, the effect of this symbiosis on GL production is unknown. Rhizobia were isolated from root nodules of Glycyrrhiza glabra, and a rhizobium that can form root nodules in G. uralensis was selected. Whole-genome analysis revealed a single circular chromosome of 6.7 Mbp. This rhizobium was classified as Mesorhizobium by phylogenetic analysis and was designated Mesorhizobium sp. J8. When G. uralensis plants grown from cuttings were inoculated with J8, root nodules formed. Shoot biomass and SPAD values of inoculated plants were significantly higher than those of uninoculated controls, and the GL content of the roots was 3.2 times that of controls. Because uninoculated plants from cuttings showed slight nodule formation, we grew plants from seeds in plant boxes filled with sterilized vermiculite, inoculated half of the seedlings with J8, and grew them with or without 100 µM KNO₃. The SPAD values of inoculated plants were significantly higher than those of uninoculated plants. Furthermore, the expression level of the CYP88D6 gene, which is a marker of GL synthesis, was 2.5 times higher than in inoculated plants. These results indicate that rhizobial symbiosis promotes both biomass and GL production in G. uralensis.     

Cell-to-cell interaction analysis of prognostic ligand-receptor pairs in human pancreatic ductal adenocarcinoma

Cell-to-cell interactions (CCIs) through ligand-receptor (LR) pairs in the tumor microenvironment underlie the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). However, there is scant knowledge of the association of CCIs with PDAC prognosis, which is critical to the identification of potential therapeutic candidates. Here, we sought to identify the LR pairs associated with PDAC patient prognosis by integrating survival analysis and single-cell CCI prediction. Via survival analysis using gene expression from cancer cohorts, we found 199 prognostic LR pairs. CCI prediction based on single-cell RNA-seq data revealed the enriched LR pairs associated with poor prognosis. Notably, the CCIs involved epithelial tumor cells, cancer-associated fibroblasts, and tumor-associated macrophages through integrin-related and ANXA1–FPR pairs. Finally, we determined that CCIs involving 33 poor-prognostic LR pairs were associated with tumor grade. Although the clinical implication of the set of LR pairs must be determined, our results may provide potential therapeutic targets in PDAC.     

Urinary and plasma proteomics to discover biomarkers for diagnosing between diabetic nephropathy and minimal change nephrotic syndrome or membranous nephropathy

The choice of treatment for primary nephrotic syndrome depends on the pathologic type of the disorder. Renal biopsy is necessary for a definitive diagnosis, but it is burdensome for the patients, and can be avoided if tests could be performed using urine or plasma. In this study, we analyzed 100 urinary proteins, 141 plasma proteins, and 57 urine/plasma ratios in cases of diabetic nephropathy (DN; n = 11), minimal change nephrotic syndrome (MCNS; n = 14), and membranous nephropathy (MN; n = 23). We found that the combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 could distinguish between MCNS and DN, with an area under the curve (AUC) of 0.9740. On the other hand, a selectivity index (SI) based on serotransferrin and immunoglobulin G, which is often used in clinical practice, distinguished them with an AUC of 0.9091. Similarly, the combination of urinary afamin and complement C3 urine/plasma ratio could distinguish between MN and DN with an AUC of 0.9842, while SI distinguished them with an AUC of 0.8538. Evidently, the candidates identified in this study were superior to the SI method. Thus, the aim was to test these biomarkers for accurate diagnosis and to greatly reduce the burden on patients.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.