Trace analysis of per- and polyfluorinated alkyl substances (PFAS) in dried blood spots - Demonstration of reproducibility and comparability to venous blood samples.

Per- and polyfluoroalkyl substances (PFAS) are a group of man-made chemicals that have been widely used in consumer, personal care, and household products for their stain- and water-repellent properties. PFAS exposure has been linked to various adverse health outcomes. Such exposure has commonly been evaluated in venous blood samples. While this sample type can be obtained from healthy adults, a less invasive method of blood collection is required when evaluating vulnerable populations. Dried blood spots (DBS) have gained attention as a biomatrix for exposure assessment given the relative ease of collection, transport, and storage. The objective of this study was to develop and validate an analytical method to measure PFAS in DBS. A workflow is presented for extracting PFAS from DBS, chemical analysis by liquid chromatography-high resolution mass spectrometry, normalization for blood mass, and blank correction to account for potential contamination. Over 80 % recovery was achieved for the 22 PFAS measured with an average coefficient of variation of 14 %. Comparison of PFAS concentrations detected in DBS and paired whole blood samples from six healthy adults was correlated (R2 > 0.9). Findings demonstrate trace levels of a broad range of PFAS in DBS can be reproducibly measured and are comparable to liquid whole blood samples. DBS can offer novel insights to environmental exposures, including during critical windows of susceptibility (i.e., in utero, early life), which have been largely uncharacterized.

Quantitative digital pathology reveals association of cell-specific PNPLA3 transcription with NAFLD disease activity.

BACKGROUND & AIMS: The I148M variant (rs738409) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is by far the most important genetic determinant of non-alcoholic fatty liver disease (NAFLD). However, in the context of NAFLD, the transcriptional regulation of PNPLA3 in human liver cells is not known. In this study, we aimed to define the relationship between PNPLA3 transcription and disease characteristics of human NAFLD. METHODS: The abundance of PNPLA3 and collagen 1α (COL1α) transcripts was quantified in situ at single-cell resolution using RNAscope® in 87 patients with NAFLD. We examined the association of PNPLA3 and COL1α transcript levels with NAFLD disease severity, defined by histology. RESULTS: While the majority of PNPLA3 transcripts were found in hepatocytes, approximately 7% of PNPLA3-positive cells co-express COL1α, representing activated myofibroblasts. There is no association between the rs738409 genotype and the level of PNPLA3 transcript. The overall PNPLA3 transcript abundance is lower in zone 1 hepatocytes, patients with higher body mass index, and those with advanced liver fibrosis. The negative association between the PNPLA3 transcript levels and liver fibrosis is largely driven by COL1α-positive cells. A significant proportion of PNPLA3 mRNA is seen in the nucleus. The cytoplasmic-to-nuclear PNPLA3 mRNA ratio is inversely associated with NAFLD disease activity. CONCLUSIONS: PNPLA3 transcript abundance and nuclear-to-cytoplasmic translocation are negatively associated with hepatic steatosis and NAFLD disease activity, while its abundance in activated myofibroblasts is inversely associated with the stage of liver fibrosis. LAY SUMMARY: A genetic variant in patatin-like phospholipase domain-containing protein 3 (or PNPLA3) is the most important genetic determinant of non-alcoholic fatty liver disease (NAFLD). However, it is not known how transcriptional regulation of the PNPLA3 gene contributes to the disease characteristics of human NAFLD. Herein, we show that the mRNA levels of PNPLA3, particularly in the cytoplasm, are negatively associated with the severity of NAFLD in humans.

Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain.

The GDA1/CD39 ecto-nucleoside triphosphate diphosphosphohydrolase (E-NTPDase) superfamily is a group of eight heavily glycosylated ecto-enzymes that hydrolyze extracellular nucleosides di- and tri-phosphates in the presence of divalent cations, to generate the monophosphate derivatives. This catalytic process differentially regulates a complex array of purinergic signaling responses. NTPDase3/CD39L3is dominantly expressed in pancreatic islet cells, where it may regulate insulin secretion, and has seven N-linked glycosylation sites with four close to five highly conserved domains called "apyrase conserved regions" (ACRs). In a manner similar to CD39, NTPDase3/CD39L3 uses ATP as its preferential substrate and also possesses significant activities toward other triphosphate and diphosphate nucleosides. To understand the mechanism of the ecto-NTPDase activity and substrate specificity, potentially impacted by N-glycans, we have generated soluble enzymatic domains of NTPDase3/CD39L3 in human embryotic kidney cells with four different glycan modifications. These include mannose5-9 glycans with kifunesine treatment, single GlcNAc-Asn by treatment with EndoH, de-glycosylated form by treatment with PNGaseF, and wild-type glycans. Our functional data indicate that the non-glycosylated NTPDase3/CD39L3 ecto-enzymatic domain retains activity, but that N-glycan attachments, such as the GlcNAc-Asn, substantially upregulate specific NTPDase activity by 2-20 fold. Both the Vmax and the Km on di- or tri-phosphate nucleosides are substantially and differentially altered by the glycan attachments. Structural modeling analysis based on putative structures derived from bacterial-originated CD39 domain proteins suggests that N-glycan modifications at Asn149 next to ACR2 and/or Asn454, N-terminal to ACR5 have critical roles in regulating the catalytic pocket of NTPDase3/CD39L3. Our data provide both new insights into the enzymatic mechanisms of NTPDase family members and further evidence that N-glycans directly modulate functional ectonucleotidase activities.