Crosstalk between oxygen signaling and iron metabolism in renal interstitial fibroblasts.

To maintain the oxygen supply, the production of red blood cells (erythrocytes) is promoted under low-oxygen conditions (hypoxia). Oxygen is carried by hemoglobin in erythrocytes, in which the majority of the essential element iron in the body is contained. Because iron metabolism is strictly controlled in a semi-closed recycling system to protect cells from oxidative stress caused by iron, hypoxia-inducible erythropoiesis is closely coordinated by regulatory systems that mobilize stored iron for hemoglobin synthesis. The erythroid growth factor erythropoietin (EPO) is mainly secreted by interstitial fibroblasts in the renal cortex, which are known as renal EPO-producing (REP) cells, and promotes erythropoiesis and iron mobilization. Intriguingly, EPO production is strongly induced by hypoxia through iron-dependent pathways in REP cells. Here, we summarize recent studies on the network mechanisms linking hypoxia-inducible EPO production, erythropoiesis and iron metabolism. Additionally, we introduce disease mechanisms related to disorders in the network mediated by REP cell functions. Furthermore, we propose future studies regarding the application of renal cells derived from the urine of kidney disease patients to investigate the molecular pathology of chronic kidney disease and develop precise and personalized medicine for kidney disease.

Predicting the anion conductivities and alkaline stabilities of anion conducting membrane polymeric materials: development of explainable machine learning models.

Anion exchange membranes (AEMs) are core components in fuel cells and water electrolyzers, which are crucial to realize a sustainable hydrogen society. The low anion conductivity and durability of AEMs have hindered the commercialization of AEM-based devices, and research and development (R&D) to improve AEM materials is often resource-intensive. Although machine learning (ML) is commonly used in many fields to accelerate R&D while reducing resource consumption, it is rarely used in the AEM field. Three problems hinder the adoption of ML models, namely, the low explainability of ML models; complication with expressing both homopolymers and copolymers in unity to train a single ML model; and difficulty in building a single ML model that comprehends various polymer types. This study presents the first ML models that solve all three problems. Our models predicted the anion conductivity for a diverse set of unseen AEM materials with high accuracy (root mean squared error = 0.014 S cm-1), regardless of their state (freshly synthesized or degraded). This enables virtual pre-synthesis screening of novel AEM materials, reducing resource consumption. Moreover, human-comprehensible prediction logic revealed new factors affecting the anion conductivity of AEM materials. Such capability to reveal new important variables for AEM materials design could shift the paradigm of AEM R&D. This proposed method is not limited to AEM materials, instead it presents a technology that is applicable to the diverse set of polymers currently available.

Gene expression changes related to bone mineralization, blood pressure and lipid metabolism in mouse kidneys after space travel.

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

Special Features of COVID-19 in the FMODB: Fragment Molecular Orbital Calculations and Interaction Energy Analysis of SARS-CoV-2-Related Proteins.

SARS-CoV-2 is the causative agent of coronavirus (known as COVID-19), the virus causing the current pandemic. There are ongoing research studies to develop effective therapeutics and vaccines against COVID-19 using various methods and many results have been published. The structure-based drug design of SARS-CoV-2-related proteins is promising, however, reliable information regarding the structural and intra- and intermolecular interactions is required. We have conducted studies based on the fragment molecular orbital (FMO) method for calculating the electronic structures of protein complexes and analyzing their quantitative molecular interactions. This enables us to extensively analyze the molecular interactions in residues or functional group units acting inside the protein complexes. Such precise interaction data are available in the FMO database (FMODB) (https://drugdesign.riken.jp/FMODB/). Since April 2020, we have performed several FMO calculations on the structures of SARS-CoV-2-related proteins registered in the Protein Data Bank. We have published the results of 681 structures, including three structural proteins and 11 nonstructural proteins, on the COVID-19 special page (as of June 8, 2021). In this paper, we describe the entire COVID-19 special page of the FMODB and discuss the calculation results for various proteins. These data not only aid the interpretation of experimentally determined structures but also the understanding of protein functions, which is useful for rational drug design for COVID-19.

Fragmentation at sp2 carbon atoms in fragment molecular orbital method.

In the fragment molecular orbital (FMO) method, a given molecular system is usually fragmented at sp3 carbon atoms. However, fragmentation at different sites sometimes becomes necessary. Hence, we propose fragmentation at sp2 carbon atoms in the FMO method. Projection operators are constructed using sp2 local orbitals. To maintain practical accuracy, it is essential to consider the three-body effect. In order to suppress the corresponding increase of computational cost, we propose approximate models considering local trimers. Numerical verification shows that the present models are as accurate as or better than the standard FMO2 method in total energy with fragmentation at sp3 carbon atoms.

High-Precision Atomic Charge Prediction for Protein Systems Using Fragment Molecular Orbital Calculation and Machine Learning.

Here, we have constructed neural network-based models that predict atomic partial charges with high accuracy at low computational cost. The models were trained using high-quality data acquired from quantum mechanics calculations using the fragment molecular orbital method. We have succeeded in obtaining highly accurate atomic partial charges for three representative molecular systems of proteins, including one large biomolecule (approx. 2000 atoms). The novelty of our approach is the ability to take into account the electronic polarization in the system, which is a system-dependent phenomenon, being important in the field of drug design. Our high-precision models are useful for the prediction of atomic partial charges and expected to be widely applicable in structure-based drug designs such as structural optimization, high-speed and high-precision docking, and molecular dynamics calculations.

Iron attenuates erythropoietin production by decreasing hypoxia-inducible transcription factor 2α concentrations in renal interstitial fibroblasts.

Iron is an essential mineral for oxygen delivery and for a variety of enzymatic activities, but excessive iron results in oxidative cytotoxicity. Because iron is primarily used in red blood cells, defective erythropoiesis caused by loss of the erythroid growth factor erythropoietin (Epo) elevates iron storage levels in serum and tissues. Here, we investigated the effects of iron in a mouse model of Epo-deficiency anemia, in which serum iron concentration was significantly elevated. We found that intraperitoneal injection of iron-dextran caused severe iron deposition in renal interstitial fibroblasts, the site of Epo production. Iron overload induced by either intraperitoneal injection or feeding decreased activity of endogenous Epo gene expression by reducing levels of hypoxia-inducible transcription factor 2α (HIF2α), the major transcriptional activator of the Epo gene. Administration of an iron-deficient diet to the anemic mice reduced serum iron to normal concentration and enhanced the ability of renal Epo production. These results demonstrate that iron overload due to Epo deficiency attenuates endogenous Epo gene expression in the kidneys. Thus, iron suppresses Epo production by reducing HIF2α concentration in renal interstitial fibroblasts.

Nuclear quantum effects of light and heavy water studied by all-electron first principles path integral simulations.

The isotopologs of liquid water, H2O, D2O, and T2O, are studied systematically by first principles PIMD simulations, in which the whole entity of the electrons and nuclei are treated quantum mechanically. The simulation results are in reasonable agreement with available experimental data on isotope effects, in particular, on the peak shift in the radial distributions of H2O and D2O and the shift in the evaporation energies. It is found that, due to differences in nuclear quantum effects, the H atoms in the OH bonds more easily access the dissociative region up to the hydrogen bond center than the D (T) atoms in the OD (OT) bonds. The accuracy and limitation in the use of the current density-functional-theory-based first principles PIMD simulations are also discussed. It is argued that the inclusion of the dispersion correction or relevant improvements in the density functionals are required for the quantitative estimation of isotope effects.

Rheumatoid nodule in the lower lip of a patient with rheumatoid arthritis: a novel case report and review of literature.

PURPOSE: Rheumatoid nodules are a well-characterized common extra-articular manifestation of rheumatoid arthritis. However, the occurrence of a rheumatoid nodule in the oral mucosa is extremely rare. PATIENTS AND METHODS: We present the case of a rheumatoid nodule in the lower lip, which rarely presents with variations in the clinical manifestation, that occurred in a 48-year-old female patient with rheumatoid arthritis. The nodule was totally excised under the clinical diagnosis of either a fibroma or salivary gland lesion. RESULTS: On histopathologic examination, within the deep mucosa, a necrobiotic nodule surrounded by elongated histiocytic cells with a focal palisaded arrangement. The lesion was diagnosed postoperatively as a rheumatoid nodule from the histopathologic and clinical findings. CONCLUSIONS: Few studies have been performed of oral rheumatoid lesions; however, a review of the published data showed that this is the first case of a rheumatoid nodule in the lower lip of a patient with rheumatoid arthritis.

The drug-specific properties of hypoxia-inducible factor-prolyl hydroxylase inhibitors in mice reveal a significant contribution of the kidney compared to the liver to erythropoietin induction.

AIMS: Kidney disease often leads to anemia due to a defect in the renal production of the erythroid growth factor erythropoietin (EPO), which is produced under the positive regulation of hypoxia-inducible transcription factors (HIFs). Chemical compounds that inhibit HIF-prolyl hydroxylases (HIF-PHs), which suppress HIFs, have been developed to reactivate renal EPO production in renal anemia patients. Currently, multiple HIF-PH inhibitors, in addition to conventional recombinant EPO reagents, are used for renal anemia treatment. This study aimed to elucidate the therapeutic mechanisms and drug-specific properties of HIF-PH inhibitors. METHODS AND KEY FINDINGS: Gene expression analyses and mass spectrometry revealed that HIF-PH inhibitors (daprodustat, enarodustat, molidustat, and vadadustat) alter Epo gene expression levels in the kidney and liver in a drug-specific manner, with different pharmacokinetics in the plasma and urine after oral administration to mice. The drug specificity revealed the dominant contribution of EPO induction in the kidneys rather than in the liver to plasma EPO levels after HIF-PH inhibitor administration. We also found that several HIF-PH inhibitors directly induce duodenal gene expression related to iron intake, while these drugs indirectly suppress hepatic hepcidin expression to mobilize stored iron for hemoglobin synthesis through induction of the EPO-erythroferrone axis. SIGNIFICANCE: Renal EPO induction is the major target of HIF-PH inhibitors for their therapeutic effects on erythropoiesis. Additionally, the drug-specific properties of HIF-PH inhibitors in EPO induction and iron metabolism have been shown in mice, providing useful information for selecting the proper HIF-PH inhibitor for each renal anemia patient.

Enhanced Coarse-Grained Molecular Dynamics Simulation with a Smoothed Hybrid Potential Using a Neural Network Model.

In all-atom (AA) molecular dynamics (MD) simulations, the rugged energy profile of the force field makes it challenging to reproduce spontaneous structural changes in biomolecules within a reasonable calculation time. Existing coarse-grained (CG) models, in which the energy profile is set to a global minimum around the initial structure, are unsuitable to explore the structural dynamics between metastable states far away from the initial structure without any bias. In this study, we developed a new hybrid potential composed of an artificial intelligence (AI) potential and minimal CG potential related to the statistical bond length and excluded volume interactions to accelerate the transition dynamics while maintaining the protein character. The AI potential is trained by energy matching using a diverse structural ensemble sampled via multicanonical (Mc) MD simulation and the corresponding AA force field energy, profile of which is smoothed by energy minimization. By applying the new methodology to chignolin and TrpCage, we showed that the AI potential can predict the AA energy with significantly high accuracy, as indicated by a correlation coefficient (R-value) between the true and predicted energies exceeding 0.89. In addition, we successfully demonstrated that CGMD simulation based on the smoothed hybrid potential can significantly enhance the transition dynamics between various metastable states while preserving protein properties compared to those obtained with conventional CGMD and AAMD.

Association Between Reduced Posterior Occlusal Contact and Alzheimer's Disease Onset in Older Japanese Adults: Results from the LIFE Study.

BACKGROUND: An association between poor oral health and cognitive decline has been reported. Most of these studies have considered the number of teeth as a criterion, only a few studies have analyzed the relationship between occlusal status and Alzheimer's disease (AD). OBJECTIVE: To elucidate whether posterior occlusal contact is associated with AD, focusing on the Eichner classification, among an older population aged 65 years or older in Japan. METHODS: This study used monthly claims data of National Health Insurance in Japan from April 2017 to March 2020. The outcome was newly diagnosed AD defined according to ICD-10 code G30. The number of teeth was estimated by dental code data, and occlusal contact was divided into three categories, namely A, B, and C, according to the Eichner classification. Multivariate Cox proportional hazards models were used to analyze the association between a new diagnosis of AD and the Eichner classification. RESULTS: A total of 22,687 participants were included, 560 of whom had newly diagnosed AD during a mean follow-up period of 12.2 months. The AD participants had a lower proportion of Eichner A and a higher proportion of Eichner C. After adjusting for covariates, hazard ratios (95% confidence intervals) with Eichner B and C were 1.34 (1.01-1.77) and 1.54 (1.03-2.30), respectively. CONCLUSION: In older people aged≥65 years old, reduced posterior occlusal contact as well as tooth loss have an impact on AD. This study emphasizes the importance of paying attention to occlusal contacts to reduce the risk of AD.

PNPO-PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity.

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.

Taxonomy of a New Parasitic Euglenid, Euglenaformis parasitica sp. nov. (Euglenales, Euglenaceae) in Ostracods and Rhabdocoels.

Parasitic euglenids have rarely been studied. We found parasitic euglenids in two species of ostracods (Cyprinotus cassidula, Dolerocypris sinensis) and two species of rhabdocoels (Mesostoma lingua, Microdalyellia armigera) in a rice field. These parasites grew and proliferated inside the host body. These parasites had pellicle strips, one emergent flagellum, and a red stigma, but no chloroplasts, and showed euglenoid movement. Inside the living host, they did not have emergent flagella and moved only by euglenoid movement, but when the host died or the parasites were isolated from the host, they extended their flagella and switched to swimming movement. We conclude that the parasites found in the four hosts that we examined are of the same species, considering the morphological characteristics and identities in the nSSU and nLSU rDNA sequences of those parasites. Molecular phylogenetic analysis showed that the parasite formed a clade with the free-living photoautotrophic species of Euglenaformis, with moderate statistical support. Therefore, the parasite is a secondary osmotroph derived from a photoautotrophic ancestor. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new species of parasitic euglenid, Euglenaformis parasitica sp. nov.

Pyridine-mediated B-B bond cleavage of tetrahydroxydiboron to synthesize n-doped SWCNTs with long-term air stability.

Neutral radicals, including carbon radicals, are highly useful chemical species for the functionalization of semiconducting materials to change their electrical and optical properties owing to their high reactivity. However, boron radicals have been limited to synthetic and reaction chemistry, with rare utilization in materials science. In this study, a mixture of tetrahydroxydiboron (B2(OH)4) and pyridine derivatives was found to act as an electron dopant for single-walled carbon nanotubes (SWCNTs) because of the electron transfer from pyridine-mediated boron radicals generated by B-B bond dissociation to neutral radicals. In particular, the radical formed from a mixture of B2(OH)4 and 4-phenylpyridine ((4-Phpy)B(OH)2·) efficiently doped electrons into the SWCNT films; thus, n-type SWCNTs with long-term air stability for more than 50 days at room temperature were prepared. Furthermore, the experimental and theoretical surface analyses revealed that the formation of stable cations from ((4-Phpy)B(OH)2·) and the efficient interaction with SWCNTs due to their high planarity served as the mechanism for their stable doping.

Drugs activating hypoxia-inducible factors correct erythropoiesis and hepcidin levels via renal EPO induction in mice.

The erythroid growth factor erythropoietin (EPO) is mainly produced by the kidneys in adult mammals and induces expansion of erythroid cells and iron use for hemoglobin synthesis. The liver also produces EPO at a lower level than the kidneys. Renal and hepatic EPO production is fundamentally regulated by hypoxia-inducible transcription factors (HIFs) in a hypoxia/anemia-inducible manner. Recently, small compounds that activate HIFs and EPO production in the kidneys by inhibiting HIF-prolyl hydroxylases (HIF-PHIs) have been launched to treat EPO-deficiency anemia in patients with kidney disease. However, the roles of the liver in the HIF-PHI-mediated induction of erythropoiesis and iron mobilization remain controversial. Here, to elucidate the liver contributions to the therapeutic effects of HIF-PHIs, genetically modified mouse lines lacking renal EPO-production ability were analyzed. In the mutant mice, HIF-PHI administration marginally increased plasma EPO concentrations and peripheral erythrocytes by inducing hepatic EPO production. The effects of HIF-PHIs on the mobilization of stored iron and on the suppression of hepatic hepcidin, an inhibitory molecule for iron release from iron-storage cells, were not observed in the mutant mice. These findings demonstrate that adequate induction of EPO mainly in the kidney is essential for achieving the full therapeutic effects of HIF-PHIs, which include hepcidin suppression. The data also show that HIF-PHIs directly induce the expression of duodenal genes related to dietary iron intake. Furthermore, hepatic EPO induction is considered to partially contribute to the erythropoietic effects of HIF-PHIs but to be insufficient to compensate for the abundant EPO induction by the kidneys.

Dietary Problems Are Associated with Frailty Status in Older People with Fewer Teeth in Japan.

This study aimed to investigate the association between dietary problems and frailty according to tooth loss in older Japanese people. This cross-sectional study included 160 older people (mean age 82.6 years) from Japan. Frailty status was assessed using the Study of Osteoporotic Fractures (SOF) criteria, which consists of (i) weight loss > 5% in the past year, (ii) inability to perform five chair stands, and (iii) self-perceived reduced energy level. Frailty was defined as the presence of ≥2 items of SOF criteria. Multivariate logistic regression analyses were performed with frailty as the dependent variable and dietary problems as the independent variable, stratified according to having <20 teeth. Low appetite and no enjoyment of eating were associated with frailty after adjusting for covariates in participants with <20 teeth. Dietary problems, including low appetite, eating alone, and negative attitudes toward enjoyment of eating were associated with a self-perceived reduced energy level in participants with <20 teeth. However, this association was not observed in participants with ≥20 teeth. In older people with fewer teeth, dietary problems have been suggested to be associated with frailty. Therefore, it may be necessary to pay attention to dietary problems, especially in older people with tooth loss.

ortho-Substituted Aryldiazonium Design for the Defect Configuration-Controlled Photoluminescent Functionalization of Chiral Single-Walled Carbon Nanotubes.

Defect functionalization of single-walled carbon nanotubes (SWCNTs) by chemical modification is a promising strategy for near-infrared photoluminescence (NIR PL) generation at >1000 nm, which has advanced telecom and bio/medical applications. The covalent attachment of molecular reagents generates sp3-carbon defects in the sp2-carbon lattice of SWCNTs with bright red-shifted PL generation. Although the positional difference between proximal sp3-carbon defects, labeled as the defect binding configuration, can dominate NIR PL properties, the defect arrangement chemistry remains unexplored. Here, aryldiazonium reagents with π-conjugated ortho-substituents (phenyl and acetylene groups) were developed to introduce molecular interactions with nanotube sidewalls into the defect-formation chemical reaction. The functionalized chiral SWCNTs selectively emitted single defect PL in the wavelength range of ∼1230-1270 nm for (6,5) tubes, indicating the formation of an atypical binding configuration, different from those exhibited by typical aryl- or alkyl-functionalized chiral tubes emitting ∼1150 nm PL. Moreover, the acetylene-based substituent design enabled PL brightening and a subsequent molecular modification of the doped sites using click chemistry.

Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.