In “cell-function editing”, the combination of biological methods with artificial methods is a promising way to effectively implement functions that live cells do not originally possess. In the present symposium review, two approaches with methodology of building “artificial organelle” were implemented for editing cellular functions. One approach is the “membrane-bound artificial organelle”, which is mainly created from polymeric nanocapsules that function in cells, and the other approach mimics the “membraneless organelle”, which has recently gained immense interest in the field of cell biology. Furthermore, some examples of artificial cells are also described, which were constructed by utilizing artificial organelles. In this context, some recent progress has been observed in the author's research on the development of polyion complex (PIC) materials, in particular, PICsome-based nanoreactors, designer coacervates for protein sequestration, and yolk-shell PIC structures that are reminiscent of artificial cells. These technologies may contribute to effective “cell editing” or “cell renovation”, which enables the edited cells to show higher performance at the target site in the human body, compared to the native cells.
One of the current critical issues in nucleic acid delivery is the efficient mRNA delivery into target cells, directed toward clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genome editing. To this end, we have developed a variety of cationic polyaspartamide derivatives with varying side chain structures because they can form nanocomplexes, termed polyplexes, with mRNA through electrostatic interactions. Interestingly, the delivery functions were highly affected by the chemical structures of the polyaspartamide side chains. Therefore, we review our previous research and provide a rationale for designing polypeptides for mRNA delivery.
Bioinspired polymeric biomaterials with excellent cytocompatibility have been designed in this study. 2-Methacryloyloxyehtyl phosphorylcholine (MPC) is a phospholipid polymer and an essential polymeric biomaterial, which has been used in various biomedical and pharmaceutical applications including implantable medical devices. Furthermore, it is a methacrylate monomer unit and can be copolymerized with other vinyl monomers via conventional radical polymerization. The water-solubility of MPC polymers depends on the molecular composition and molecular weight of the polymers. PMB is a water-soluble polymer copolymerized with hydrophobic n-butyl methacrylate, and can be used as a solubilizing agent for poorly soluble drugs. The phospholipid polymers showed low cytotoxicity, and the solubilized drugs effectively not only penetrated into the cells but also into the surrounding tissues. In addition, the water-soluble MPC polymer containing a phenylboronic acid moiety was observed to spontaneously form polymeric hydrogels with polyol compounds. The reversible polymer hydrogels were used as artificial extracellular matrices for cell immobilization and cell engineering. Polymeric biomaterials with intelligent interfaces might be explored as innovative techniques for application in pharmaceutical and life sciences.
Remarkable progress in our ability to analyze diseased tissue has revolutionized our understanding of disease. From a simplistic understanding of abnormalities in bulk tissue, there is now increasing recognition that the heterogeneous and dynamically evolving disease microenvironment plays a crucial role in disease pathogenesis and progression as well as in the determination of therapeutic response. The disease microenvironment consists of multiple cell types as well as the various factors that these cells secrete. There is now immense interest in treatment strategies that target or modify the abnormal disease microenvironment, and a deeper understanding of the mechanisms that drive the formation, maintenance, and progression of the disease microenvironment is thus necessary. The advent of 3-dimensional (3D) cell culture technology has made possible the reconstitution of the disease microenvironment to a previously unimaginable extent in vitro. As an intermediate between traditional in vitro models based on 2-dimensional (2D) cell culture and in vivo models, 3D models of disease enable the in vitro reconstitution of complex interactions within the disease microenvironment which were unamenable in 2D while simultaneously allowing the mechanistic analysis of these interactions that would be difficult to perform in vivo. This symposium review aims to highlight the promise of using 3D cell culture technology to model and analyze the disease microenvironment using pancreatic cancer as an example.
Vigorous efforts are being made to manipulate cellular functions in a desirable manner for biomedical purposes. Recent advances in platform technologies have made cell editing achievable; this includes generation of induced pluripotent stem cells and chimeric antigen receptor T cells, as well as direct cell reprogramming. mRNA, as compared to DNA, is an excellent tool for potentiating cell editing technologies, owing to its distinct properties in gene introduction. Herein, hepatocytes were edited ex vivo and in vivo, by introducing pro-survival mRNA, to be resistant to cell death. DNA-based introduction of pro-survival gene poses safety concerns due to its genomic integration, as prolonged and uncontrolled expression of pro-survival proteins after the integration may promote cancer. In contrast, mRNA lacks such a risk. Moreover, mRNA-based introduction of Bcl-2, a pro-survival factor, was more effective in preventing the death of cultured hepatocytes than Bcl-2 plasmid DNA (pDNA) introduction. Mechanistically, mRNA induced protein expression in a larger percentage of the hepatocytes compared to pDNA, presumably because the process of pDNA nuclear entry in transfection is challenging. In hepatocyte transplantation to mouse liver, ex vivo introduction of Bcl-2 mRNA significantly improved the engraftment efficiency of the hepatocytes, leading to successful functional support of the liver in a mouse model of chronic hepatitis. Furthermore, in vivo administration of Bcl-2 mRNA exhibited an anti-apoptotic effect on the hepatocytes of a mouse model of fulminant hepatitis. These results demonstrate the potential advantages of mRNA introduction over DNA introduction in cell editing.
Although the concept of a drug delivery system (DDS) is usually applied to conventional drug therapy, it is also important for cell-based therapy. The surface manipulation of living cells represents a powerful tool for controlling cell behaviors in the body, such as enhancement of cell-cell interactions, targeted delivery of cells, and protection from immunological rejection. Functional groups, including amines, thiols, and carbonyls, offer excellent opportunities for chemical modification through the formation of covalent bonds with exogenous molecules. Non-natural reactive groups introduced by metabolic labeling were recently utilized for targeted chemical modification. On the other hand, noncovalent strategies are also available; two major examples are electrostatic interaction with a negative charge on the cell surface and hydrophobic insertion or interaction with the cell membrane. In this study, we analyzed factors affecting cell surface modifications using PEG-lipid and succeeded in enhancing the efficacy of modification by cyclodextrin. Then, mesenchymal stem cells (MSCs), whose therapeutic effect has been demonstrated at the clinical stage and which have been clinically used as a drug, were decorated with PEG-lipid conjugates having a targeted ligand such as peptide or scFv, which are recognized by ICAM1. The peptide or scFv decoration enhanced the cell adhesion of MSCs on cytokine treated-endothelial cells. This technique will prompt the targeted delivery of MSCs to intended therapy sites, and underscores the promise of cell surface engineering as a tool for improving cell-based therapy.
Vitamin K is a fat-soluble vitamin that plays an important role in blood coagulation and bone formation. Vitamin K has homologues due to differences in the side chain structure, phylloquinone (abbreviated as vitamin K1, PK) having a phytyl side chain and menaquinones (MK-n, n=1 to 14) having an isoprenoid side chain structure. The main vitamin K that we take from our daily diet is PK, and a fermented food, natto, contains MK-7 produced by Bacillus subtilis natto. However, the majority of vitamin K present in the tissues of mammals, including humans, is menaquinone-4 (abbreviated as vitamin K2, MK-4) having a geranylgeranyl side chain. This reason is that PK or MK-n obtained in the diet is converted into MK-4 in the body. We identified that the UbiA prenyltransferase domain containing protein 1 (UBIAD1) is the conversion enzyme of PK and MK-n to MK-4. The physiological roles of MK-4 in all tissues of the whole body and the physiological significance of MK-4 converted from PK and MK-n by UBIAD1 have not been sufficiently elucidated yet. To investigate the function of UBIAD1 in vivo, we generated UBIAD1 systemic knockout mice and tissue-specific UBIAD1 knockout mice. In this paper, we introduce the usefulness of vitamin K for diseases that may involve vitamin K and UBIAD1.
trans-Fatty acids (TFAs), including elaidic acid and linoelaidic acid, are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. TFAs are not synthesized in the human body, but are taken into the body from various foods, which are mainly produced during industrial food manufacturing. Recent epidemiological studies have revealed that TFA consumption is a major risk factor for various disorders, such as atherosclerosis, cardiovascular diseases, allergic diseases, and dementia. However, the underlying pathogenic mechanisms of TFA-related disorders and the specific molecular targets evoking TFA toxicity are largely unknown. To elucidate the molecular mechanisms by which TFAs cause the cytotoxicity, we focused on cell death and inflammation, which are the main and common pathogenesis of the TFA-related diseases, and analyzed the effects of TFAs on cellular responses to various stimulations inducing cell death and inflammation. This review provides recent progress in our studies on the molecular mechanisms causing toxic actions of TFAs, which lead to diverse TFA-related disorders.
The intestine is exposed to a variety of exogenous materials that are harmful, harmless, or useful, such as pathogenic viruses and bacteria, intestinal bacteria, or food components. As such, the intestinal immune system is important for the regulation of immunological homeostasis and biological defense. Accumulating evidence indicates that gut environmental factors, such as dietary components and intestinal bacteria are critical for controlling intestinal immunity, and thereby, health and disease. Among the important dietary components are fatty acids, which are metabolized to lipid mediators that act as signaling molecules and regulate immune responses. In previous work, we identified lipid mediators derived from ω3 fatty acids, such as 17,18-epoxyeicosatetraenoic acid, 15-hydroxyeicosapentaenoic acid, and 14-hydroxydocosapentaenoic acid, which show potent anti-allergic and anti-inflammatory activities. In addition, we revealed that lipid mediators play key roles in the enhancement of intestinal Immunoglobulin A responses, which provide the first line of defense against viral and bacterial infectious diseases. Here, we review the anti-allergic, anti-inflammatory, and host-protective effects of lipid mediators mainly derived from dietary lipids.
Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Animals must be able to metabolize the various selenocompounds in meat, fish and vegetables to utilize Se for selenoprotein synthesis. It is known that the biological, nutritional, and toxicological effects of Se are strongly dependent on its chemical form. First, we evaluated the nutritional availability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo. Second, we evaluated that gut microflora might contributes to the Se nutritional availability. Se is mainly excreted into urine. However, a substantial amount of Se was secreted into bile although Se was hardly detected in feces. Third, we evaluated the biological significance of biliary secretion of Se in terms of mineral nutrition. Finally, we discussed the entire Se metabolism in gut contributing to Se homeostasis in animal.
Cellular transport systems for both essential and toxic trace elements remain elusive. In our studies on the transport systems for cadmium (Cd), we found that the cellular uptake of Cd is mediated by the transporter for manganese (Mn). We identified ZIP8 and ZIP14, members of the ZIP zinc (Zn) transporter family, as transporters having high affinities for both Cd and Mn. Notably, the uptake of Cd into rice root from soil is mediated by a transporter for Mn as well. We found that ZIP8 is highly expressed at the S3 segment of the kidney proximal tubule and can transport glomerulus-filtered Cd and Mn ions in the lumen into epithelial cells of the proximal tubule, suggesting that ZIP8 has an important role in the renal reabsorption of both toxic Cd and essential Mn. Mutations in ZIP8 and ZIP14 genes were found in humans having congenital disorders associated with the disturbed transport of Mn, although ZIP8 mutation causes whole-body Mn deficiency while ZIP14 mutation causes Mn accumulation in the brain. Mutations in ZnT10, a Zn transporter responsible for Mn excretion, also cause hyperaccumulation of Mn in the brain. Results of genome-wide association studies have indicated that ZIP8 SNPs are involved in a variety of common diseases. Thus, ZIP8, ZIP14, and ZnT10 play crucial roles in the transport of Mn and thereby control Mn- and Cd-related biological events in the body.
Na ionophores increase intracellular Na+ ([Na+]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10−6-3×10−5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10−5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (If), and it increased the transient outward potassium current (Ito) in Purkinje cells. In addition, monensin decreased the sodium current (INa), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (ICa) in ventricular myocytes. The Na+-Ca2+ exchange current (INa-Ca) was augmented particularly in the reverse mode, and the Na+-K+ pump current (INa-K) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (IK,ATP) could be induced by monensin. Notably, the inward rectifying K+ current (IK1), and the slow delayed outward K+ current (IKs) were not affected evidently by monensin. Collectively, alteration of [Na+]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na+]i should be taken into consideration in the action of drugs affecting [Na+]i such as digitalis, Na+ channel blockers, and Na+ channel activating agents.
Fungi are eukaryotic microorganisms that show complex life cycles, including both anamorph and teleomorph stages. Beta-1,3-1,6-glucans (BGs) are major cell wall components in fungi. BGs are also found in a soluble form and are secreted by fungal cells. Studies of fungal BGs extensively expanded from 1960 to 1990 due to their applications in cancer immunotherapy. However, progress in this field slowed down due to the low efficacy of such therapies. In the early 21st century, the discovery of C-type lectin receptors significantly enhanced the molecular understanding of innate immunity. Moreover, pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) were also discovered. Soon, dectin-1 was identified as the PRR of BGs, whereas BGs were established as PAMPs. Then, studies on fungal BGs focused on their participation in the development of deep-seated mycoses and on their role as a source of functional foods. Fungal BGs may have numerous and complex linkages, making it difficult to systematize them even at the primary structure level. Moreover, elucidating the structure of BGs is largely hindered by the multiplicity of genes involved in cell wall biosynthesis, including those for BGs, and by fungal diversity. The present review mainly focused on the characteristics of fungal BGs from the viewpoint of structure and immunological activities.
In 2010, the in-hospital practical training period for pharmacy students was extended from 4 to 11 weeks. We have conducted questionnaire surveys of these students every year with the aim of reviewing the quality of training by conduction of surveys and evaluations. However, it was not clear whether reviewing based on the questionnaire results improved student satisfaction with the in-hospital practical training. Therefore, the aim of this study was to verify the validity of reviewing based on the questionnaire results by analyzing the data accumulated during the long-term practical training. A questionnaire survey was conducted of 333 5th-year students upon completion of practical training at Chiba University Hospital from 2010 to 2017. Students self-evaluated their attitude toward practical training on a 6-point scale and their satisfaction level for each component of the practical training on a 5-point scale. The students were also allowed to share their feelings about hospital pharmacy work. Repeated review of the training content can facilitate communication with patients, which was lacking at the beginning of the training period. Improved communication led to higher-quality pharmacy practice and increased student satisfaction. Meanwhile, changes to work procedures may reduce student satisfaction unless the training strategy is reviewed accordingly. Because the work of hospital pharmacists is constantly changing, it is considered that the content of the practical training should be revised accordingly through continuous conduction of surveys and evaluations, thereby enabling optimal practical training.
Dosage of pharmaceuticals is determined and approved based on pivotal clinical trial results in Phase 3. However, in the post-marketing setting, it is often adjusted according to conditions of individual patients. Some pharmaceuticals are used at a lower dose than the approved dose for safety reasons or in elderly patients. In this study, we examined the relationships between dose discontinuation or dose reduction, for safety reasons as well as participation of elderly patients in clinical trials, and lower-dose prescriptions in the post-marketing setting. We collated the dataset of 113 eligible pharmaceuticals from those that were approved in Japan between 2005 and 2014. Subsequently, we calculated the proportions of patients who withdrew from the study, whose medication was discontinued, or dose reduced due to adverse events, and of elderly patients (over sixty-five years old) to those who were exposed to the approved dose range in the pivotal clinical trials. Then their relationships with lower-dose prescription in the post-marketing setting were examined using Mann-Whitney U test. The proportions of patients who withdrew from the study (p=0.0019), whose medication was discontinued owing to adverse events (p=0.0007), or whose dose was reduced owing to adverse events (p<0.0001) were significantly higher for “lower-dose prescription drugs” than those for other drugs; however, the proportion of elderly patients did not show this significant increase in the “lower-dose prescription drugs” group.
Medication non-adherence in the elderly population is a major problem, preventing them from obtaining optimal therapeutic effects. Identifying the factors affecting medication adherence is crucial for improving and maintaining health among the elderly population and enhance healthcare economy. The purpose of this study was to examine the prevalence of self-reported medication adherence, and identify the associated factors and the influence of health-related quality of life (HRQOL) in the Japanese community-dwelling elderly population. This cross-sectional study was part of the Nakajima study and targeted inhabitants aged ≥60 years who underwent health examinations in 2017. Data regarding medication adherence were acquired through interviews and self-administered questionnaires. Medication adherence were assessed using a visual analog scale, and HRQOL was assessed by EuroQol five-dimensional questionnaire with 3 levels. Among the 455 participants, low and high medication adherence were seen in 9.7% and 66.2% of the participants, respectively (visual analog scores <80% and ≥95%, respectively). Medication adherence was significantly lower in participants taking medications ≥3 times daily than in those taking medications once or twice daily; a regimen involving drug administration ≥3 times daily had significantly lower odds of medication adherence. The use of a drug profile book and HRQOL had significant positive association with medication adherence. Our results suggest that low dosing frequency and using a drug profile book was positively associated with medication adherence among elderly persons, which in turn could enhance their QOL.
The aims of the present study were to investigate the effects of fenofibrate and bezafibrate on the risk of development of diabetic retinopathy (DR) in patients with type 2 diabetes and dyslipidemia. Japanese working age patients with type 2 diabetes and dyslipidemia were extracted from the Nihon University School of Medicine Clinical Data Warehouse. These patients were divided into three groups: control group (n=2549), fenofibrate group (n=40), and bezafibrate group (n=135). Multivariate logistic regression analysis was performed to assess the association between fibrates and the development of DR. After adjustment for covariates, fenofibrate showed no association with the risk of DR [adjusted odds ratio (OR), 0.160; 95% CI, 0.021-1.209; p=0.0758]. Bezafibrate also showed no association with the risk of DR (adjusted OR, 0.731; 95% CI, 0.411-1.299; p=0.2855). However, poor control of hemoglobin A1c (HbA1c ≥8.0%; adjusted OR, 3.623; 95% CI, 2.649-4.956; p<0.0001) and high low-density lipoprotein cholesterol (LDL-C ≥140 mg/dL; adjusted OR, 1.399; 95% CI, 1.013-1.932; p=0.0415) within the follow-up period of type 2 diabetes and dyslipidemia increased the risk of DR. Our results suggested that to prevent development of DR in patients with type 2 diabetes and dyslipidemia, controlling LDL-C levels as well as HbA1c levels under coexistence type 2 diabetes and dyslipidemia is more important than the selection of fibrate.
This study investigated the effects of fenofibrate and bezafibrate on risk of diabetic retinopathy (DR) in Japanese working age patients with type 2 diabetes and dyslipidemia using electronic medical records. Although these fibrates did not affect development of DR, poor control of hemoglobin A1c (HbA1c) and low-density lipoprotein cholesterol (LDL-C) within the follow-up period increased the risk of DR. Thus, controlling HbA1c and LDL-C levels under coexistence type 2 diabetes and dyslipidemia is more important than the selection of fibrate.