Relationship between Adsorption and Toxicity of Nephrotoxic Drugs in Microphysiological Systems (MPS).

Microphysiological systems (MPS) are an emerging technology for next-generation drug screening in non-clinical tests. Microphysiological systems are microfluidic devices that reconstitute the physiological functions of a human organ using a three-dimensional in vivo-mimicking microenvironment. In the future, MPSs are expected to reduce the number of animal experiments, improve prediction methods for drug efficacy in clinical settings, and reduce the costs of drug discovery. However, drug adsorption onto the polymers used in an MPS is a critical issue for assessment because it changes the concentration of the drug. Polydimethylsiloxane (PDMS), a basic material used for the fabrication of MPS, strongly adsorbs hydrophobic drugs. As a substitute for PDMS, cyclo-olefin polymer (COP) has emerged as an attractive material for low-adsorption MPS. However, it has difficulty bonding with different materials and, therefore, is not commonly used. In this study, we assessed the drug adsorption properties of each material constituting an MPS and subsequent changes in drug toxicity for the development of a low-adsorption MPSs using COP. The hydrophobic drug cyclosporine A showed an affinity for PDMS and induced lower cytotoxicity in PDMS-MPS but not in COP-MPS, whereas adhesive tapes used for bonding adsorbed a significant quantity of drugs, lowering their availability, and was cytotoxic. Therefore, easily-adsorbed hydrophobic drugs and bonding materials having lower cytotoxicity should be used with a low-adsorption polymer such as COP.

Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule.

Of late, numerous microphysiological systems have been employed to model the renal proximal tubule. Yet there is lack of research on refining the functions of the proximal tubule epithelial layer-selective filtration and reabsorption. In this report, pseudo proximal tubule cells extracted from human-induced pluripotent stem cell-derived kidney organoids are combined and cultured with immortalized proximal tubule cells. It is shown that the cocultured tissue is an impervious epithelium that offers improved levels of certain transporters, extracellular matrix proteins collagen and laminin, and superior glucose transport and P-glycoprotein activity. mRNA expression levels higher than those obtained from each cell type were detected, suggesting an anomalous synergistic crosstalk between the two. Alongside, the improvements in morphological characteristics and performance of the immortalized proximal tubule tissue layer exposed, upon maturation, to human umbilical vein endothelial cells are thoroughly quantified and compared. Glucose and albumin reabsorption, as well as xenobiotic efflux rates through P-glycoprotein were all improved. The data presented abreast highlight the advantages of the cocultured epithelial layer and the non-iPSC-based bilayer. The in vitro models presented herein can be helpful in personalized nephrotoxicity studies.

Boyden chamber-based compartmentalized tumor spheroid culture system to implement localized anticancer drug treatment.

In anticancer drug development, it is important to simultaneously evaluate both the effect of drugs on cell proliferation and their ability to penetrate tissues. To realize such an evaluation process, here, we present a compartmentalized tumor spheroid culture system utilizing a thin membrane with a through-hole to conduct localized anticancer treatment of tumor spheroids and monitor spheroid dimensions as an indicator of cell proliferation. The system is based on a commercialized Boyden chamber plate; a through-hole was bored through a porous membrane of the chamber, and the pre-existing 0.4 µm membrane pores were filled with parylene C. A HepG2 spheroid was immobilized onto the through-hole, separating the upper and lower compartments. Fluorescein (to verify the isolation between the compartments) and tirapazamine (TPZ; to treat only the lower part of the spheroid) were added to the upper and lower compartments, respectively. Since the transportation of fluorescein was blocked during treatment, i.e., the upper and lower compartments were isolated, it was confirmed that localized TPZ treatment was successfully conducted using the developed system. The effect of localized TPZ treatment on cell proliferation was estimated by measuring the maximum horizontal cross-sectional areas in the upper and lower parts of the spheroid by microscopic observations. This system can, thus, be used to perform localized anticancer drug treatment of tumor spheroids and evaluate the effect of drugs on cell proliferation.

Medical phycology 2017.

In 2014, ISHAM formed a new working group: "Medical Phycology: Protothecosis and Chlorellosis." The purpose of this working group is to help facilitate collaboration and communication among people interested in the pathogenic algae, to share ideas and work together. Here we present reports on recent work we have done in five areas. 1. The history of medical phycology as a branch of science. 2. Aspects of the genetics of Prototheca. 3. Aspects of the proteins of Prototheca. 4. Human infections caused by Prototheca. 5. Dairy cow mastitis caused by Prototheca.

Synthesis and herbicidal activity of 3-{[(hetero)aryl]methanesulfonyl}-4,5-dihydro-1,2-oxazole derivative; Discovery of the novel pre-emergence herbicide pyroxasulfone.

Thiocarbamate sulfoxides, which are the active forms of thiocarbamate herbicides, are quickly conjugated with glutathione and decomposed in soil. To achieve more potent and stable herbicidal activity, we previously developed a 5-{[(2,6-difluorophenyl)methoxy]methyl}-5-methyl derivative, which has a 4,5-dihydro-1,2-oxazole ring in place of the thiocarbamate to prevent conjugation and decomposition. Although the derivative showed pre-emergence herbicidal activity under flooded conditions, it displayed no herbicidal activity under upland conditions. In contrast, a 5-(methoxymethyl)-5-methyl derivative showed pre-emergence herbicidal activity against grass weeds under upland conditions. The aim of this study was to obtain a more potent compound with improved physicochemical properties for use as a pre-emergence upland herbicide via the structural optimization of a 3-{[(hetero)aryl]methanesulfonyl}-4,5-dihydro-1,2-oxazole as the core structure. In this way, we have developed the pre-emergence herbicide 3-{[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]methanesulfonyl}-5,5-dimethyl-4,5-dihydro-1,2-oxazole, which has been named "pyroxasulfone." This novel compound displayed excellent herbicidal activity against grass and broadleaf weeds under upland conditions with no phytotoxicity against crops.

Potential of carotenoids in aquatic yeasts as a phylogenetically reliable marker and natural colorant for aquaculture.

Apart from Xanthophyllomyces dendrorhous, pink colony-forming yeasts have not been examined as a pigmentation source in captive animals. In this study, aquatic yeasts were screened with a view to abundances of carotenoids. Phylogenetic analyses of these caroetnoid-rich yeasts based on large subunit ribosomal RNA gene (LSU rDNA) partial sequences showed that all belonged to the order Sporidiobolales. Both the qualitative and the quantitative differences in carotenoids between the yeasts appeared to be consistent with their phylogenetic affiliations. This information might be useful in the selection of pigment-rich yeasts containing specific carotenoids from a large number of strains. We also found, for the first time, the potential of a pigment-rich Rhodotorula strain as a colorant for aquaculture. The integuments of tilapia and carp fed the alkali-treated cells of strain Rhodotorula dairenensis Sag 17 were pigmented after 3 months of cultivation. The fish integuments retained the yeast carotenes shortly after the start of feeding, and were converted to the fish-specific xanthophylls in vivo.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

Repeated batch cultivation of the hydrocarbon-degrading, micro-algal strain Prototheca zopfii RND16 immobilized in polyurethane foam.

This study reports on the stability of the cells of a heterotrophic green micro-algal strain Prototheca zopfii RND16 immobilized in polyurethane foam (PUF) cubes during degradation of mixed hydrocarbon substrate, which was composed of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), in 5 successive cycles of repeated batch cultivation at 30 degrees C. Both RND16 cells and mixed hydrocarbon substrate components had been entrapped in PUF cubes through cultivation. PUF-immobilized RND16 degraded n-alkanes almost completely, whereas the strain hardly degraded PAHs in PUFs, rather they accumulated in the matrices. It is noteworthy that this result is strikingly different from that of the free-living cell culture, where RND16 reduced concentrations of both n-alkanes and PAHs. However, PAHs accumulation in the PUFs did not impair the performance of the immobilized alga to utilize n-alkanes. These results suggest that the PUFs harboring RND16 cells could be used repeatedly for selective retrieval of PAHs from oil-polluted waters after preferential biodegradation of n-alkanes by algae.

Direct evidence for redundant segmental replacement between multiple 18S rRNA genes in a single Prototheca strain.

Informational genes such as those encoding rRNAs are related to transcription and translation, and are thus considered to be rarely subject to lateral gene transfer (LGT) between different organisms, compared to operational genes having metabolic functions. However, several lines of evidence have suggested or confirmed the occurrence of LGT of DNA segments encoding evolutionarily variable regions of rRNA genes between different organisms. In the present paper, we show, for the first time to our knowledge, that variable regions of the 18S rRNA gene are segmentally replaced by multiple copies of different sequences in a single strain of the green microalga Prototheca wickerhamii, resulting in at least 17 genotypes, nine of which were actually transcribed. Recombination between different 18S rRNA genes occurred in seven out of eight variable regions (V1-V5 and V7-V9) of eukaryotic small subunit (SSU) rRNAs. While no recombination was observed in V1, one to three different recombination loci were demonstrated for the other regions. Such segmental replacement was also implicated for helix H37, which is defined as V6 of prokaryotic SSU rRNAs. Our observations provide direct evidence for redundant recombination of an informational gene, which encodes a component of mature ribosomes, in a single strain of one organism.

Phylogeny of the non-photosynthetic green micro-algal genus Prototheca (Trebouxiophyceae, Chlorophyta) and related taxa inferred from SSU and LSU ribosomal DNA partial sequence data.

All five species in the heterotrophic micro-algal genus Prototheca and their relatives were compared for the extent of nucleotide divergence in the nuclear small-subunit (SSU) and in the 5' end of large-subunit (LSU) ribosomal RNA genes (rDNAs). Phylogenetic analysis based on combined SSU and LSU rDNA sequence alignment was implemented with the neighbor-joining, the maximum-parsimony, and the maximum-likelihood methods. The relationships among the species of Prototheca based on this data set were largely concordant with those inferred from SSU or LSU rDNA sequences alone. The obtained phylogenetic trees indicated that P. stagnora and P. ulmea should be regarded as different species and that both of the species as well as P. moriformis were placed in a cluster represented by P. zopfii, whereas P. wickerhamii was not directly grouped together with the other members of Prototheca and was more closely related to the autotrophic alga Auxenochlorella protothecoides. Therefore, the genus Prototheca is paraphyletic in its present circumscription; and these conclusions lead us to propose the transfer of P. wickerhamii to Auxenochlorella or to a new genus. On the basis of nucleotide sequence similarities, unlike SSU rDNA, the LSU rDNA region examined in this study appeared to be variable in recognizing a heterogeneity within a single species P. zopfii, which had been shown earlier in a chemotaxonomic study.

Isolation, characterization, and fermentative pattern of a novel thermotolerant Prototheca zopfii var. hydrocarbonea strain producing ethanol and CO2 from glucose at 40 degrees C.

A novel thermotolerant strain of the achlorophyllous micro-alga Prototheca was isolated from a hot spring. The isolate was found to produce an appreciable amount of ethanol and CO2 from glucose under anoxic conditions at both 25 and 40 degrees C; this type of alcohol fermentation has not yet been reported in the genus Prototheca. Moreover, it also evolved gas from sucrose after a time lag at 40 degrees C. Its taxonomic characteristics coincided with those of Prototheca zopfii var. hydrocarbonea, and phylogenetic analysis, based on a small-subunit (SSU) rDNA sequence, also revealed a close relationship between the two strains. D-lactic acid, ethanol, CO2 and a trace of acetic acid were produced from glucose, but L-lactic acid, formic acid, and H2 were not. At 25 degrees C, D-lactic acid and ethanol were produced in approximately equimolar amounts under N2/H2/CO2, whereas ethanol production was predominant under N2. More ethanol was produced at 40 degrees C than at 25 degrees C irrespective of the gas composition in the atmosphere. This is the first report on gas production from glucose and on the changes in the fermentative patterns as a function of temperature for the genus Prototheca.

Optimization of heterotrophic culture conditions for n-alkane utilization and phylogenetic position based on the 18S rDNA sequence of a thermotolerant Prototheca zopfii strain.

This study reports on the optimization of the culture conditions for a thermotolerant eukaryotic algal strain, Prototheca zopfii RND16, which can effectively degrade and assimilate n-alkanes at elevated temperatures. RND16 was able to grow on 1% (v/v) n-alkanes (C14-C17) at temperatures up to 38 degrees C. This ability differs from a previous finding that P. zopfii did not grow on hydrocarbons under temperature conditions above 25 degrees C. Increasing the temperature from 25 degrees C to 30-35 degrees C resulted in an increase in the rate of n-alkanes consumption during growth of RND16 in quiescent culture. In shaking culture at 35 degrees C, RND16 removed a 1% n-alkanes mixture almost completely at the basal medium salinity within 8 d. However, an appreciable decrease in the extent of hydrocarbon utilization was observed with increasing salinity and substrate concentration in the medium. The slow consumption of the n-alkanes in the early stage of degradation at 25 degrees C was improved by supplementing 0.1% glucose. A comparative study on the nuclear small subunit rDNA (18S rDNA) sequences of three strains within the genus Prototheca revealed that both P. zopfii ATCC 30253, which utilize n-alkanes at room temperature and P. moriformis ATCC 50081, which does not assimilate n-alkanes, are closely related to RND16.

Bioremediation of fish cannery wastewater with yeasts isolated from a drainage canal.

Several yeasts were isolated from a drainage canal in a Japanese fish food processing factory. They were characterized by the decomposition of organic polymers such as proteins and reducing sugars, their growth in the wastewater, the decrease in total organic carbon (TOC), and taxonomy. Three strains of yeast dominated the sample: Debaryomyces occidentalis (P1), Trichosporon ovoides (P19), and a strain that could not be identified (S27). Strain P19 had the highest TOC-decreasing activity and was immobilized onto chitosan beads. The immobilized yeasts reduced the TOC from 1.2 x 10(3) to 3.0 x 10(2) mg of C/L per day in the fish cannery wastewater.