Evaluation of the applicability of GARDskin to predict skin sensitizers in extracts from medical device materials.

Biocompatibility testing of medical devices is governed by the ISO 10993 series of standards and includes evaluation of skin sensitization potential of the final product. A majority of all medical devices are tested using in vivo methods, largely due to the lack of in vitro methods validated within the applicability domain of solid materials. The GARDskin method for assessment of chemical skin sensitizers is a validated method included in the OECD Test Guideline 442E, based on evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature in a dendritic cell-like cell, following test chemical exposure. The current study aimed to evaluate the applicability of GARDskin for the purpose of testing solid materials by incorporation of extraction procedures described in ISO 10993-12:2021, as well as to demonstrate the functionality of the proposed protocols, by testing of custom-made materials spiked with sensitizing agents. It was shown that GARDskin is compatible with both polar and non-polar extraction vehicles frequently used for the purpose of medical device biological testing. Further, exploring three different material types spiked with up to four different sensitizing agents, as well as three unspiked control materials and commercial reference products, it was shown that the method correctly classified all evaluated test materials. Taken together, the data presented suggest that GARDskin may constitute a valid alternative to in vivo experimentation for the purpose of skin sensitization assessment of medical devices.

Biocompatibility of a new PD solution for Japan, Reguneal™, measured as in vitro proliferation of fibroblasts.

BACKGROUND: The aim of this study was to investigate in vitro biocompatibility of Reguneal™, a new bicarbonate containing peritoneal dialysis fluid (PDF) for Japan, and compare it with other PDFs available in that country. METHODS: We assessed basal cytotoxicity using in vitro proliferation of cultured fibroblasts, L-929, determining the quantity of living cells by the uptake of Neutral Red. Levels of ten glucose degradation products (GDPs) were measured by a validated ultrahigh-performance liquid chromatography method in combination with an ultraviolet detector. We compared inhibition of fibroblast cell growth between brands of PDF, adjusting for dextrose and GDP concentrations using random-effects mixed models. RESULTS: The results demonstrate that cytotoxicity of Reguneal™ is comparable to a sterile-filtered control and is less cytotoxic than most of the other PDFs, most of which significantly inhibited cell growth. As a "class effect", increasing dextrose and GDP concentrations were non-significantly but positively associated with cytotoxicity. As a "brand effect", these relationships varied widely between brands, and some PDFs had significant residual effects on basal cytotoxicity through mechanisms that were unassociated with either dextrose or GDP concentration. CONCLUSION: Our study suggests that Reguneal™ is a biocompatible PDF. The results of our study also highlight that dextrose and GDPs are important for biocompatibility, but alone are not a complete surrogate. The results of our study need to be confirmed in other tissue culture models, and should lead to further research on determinants of biocompatibility and the effect of such PDFs on clinical outcomes.

Identification of novel substrates and structure-activity relationship of cellular uptake mediated by human organic cation transporters 1 and 2.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP.

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli.

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.