Adeno-associated virus type 2 in US children with acute severe hepatitis.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Design considerations for open-well microfluidic platforms for hypoxic cell studies.

Regions of hypoxia are common in solid tumors and are associated with enhanced malignancy, metastasis, and chemo/radio resistance. Real-time hypoxic cellular experimentation is challenging due to the constant need for oxygen control. Most microfluidic platforms developed thus far for hypoxic cell studies are burdened by complex design parameters and are difficult to use for uninitiated investigators. However, open-well microfluidic platforms enable short and long term hypoxic cell studies with an ease of use workflow. Specifically, open-well platforms enable manipulation and addition of cells, media, and reagents using a micropipette for hypoxic cell studies in tunable dissolved oxygen concentrations as low 0.3 mg/l. We analyzed design considerations for open-well microfluidic platforms such as media height, membrane thickness, and impermeable barriers to determine their effects on the amount of dissolved oxygen within the platform. The oxygen concentration was determined by experimental measurements and computational simulations. To examine cell behavior under controlled oxygen conditions, hypoxia-induced changes to hypoxia inducible factor activity and the mitochondrial redox environment were studied. A fluorescent reporter construct was used to monitor the stabilization of hypoxia inducible factors 1α and 2α throughout chronic hypoxia. Reporter construct fluorescence intensity inversely correlated with dissolved oxygen in the medium, as expected. Additionally, the glutathione redox poise of the mitochondrial matrix in living cancer cells was monitored throughout acute hypoxia with a genetically encoded redox probe and was observed to undergo a reductive response to hypoxia. Overall, these studies validate an easy to use open-well platform suitable for studying complex cell behaviors in hypoxia.

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (EGSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (ß-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and ß-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1-independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of ß-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while ß-lap elicits off-target responses at its effective doses.

Methods to study the tumor microenvironment under controlled oxygen conditions.

The tumor microenvironment (TME) is a complex heterogeneous assembly composed of a variety of cell types and physical features. One such feature, hypoxia, is associated with metabolic reprogramming, the epithelial-mesenchymal transition, and therapeutic resistance. Many questions remain regarding the effects of hypoxia on these outcomes; however, only a few experimental methods enable both precise control over oxygen concentration and real-time imaging of cell behavior. Recent efforts with microfluidic platforms offer a promising solution to these limitations. In this review, we discuss conventional methods and tools used to control oxygen concentration for cell studies, and then highlight recent advances in microfluidic-based approaches for controlling oxygen in engineered platforms.

Förster resonance energy transfer-based sensor targeting endoplasmic reticulum reveals highly oxidative environment.

The glutathione thiol/disulfide couple is the major redox buffer in the endoplasmic reticulum (ER); however, mechanisms by which it contributes to the tightly regulated redox environment of this intracellular organelle are poorly understood. The recent development of genetically encoded, ratiometric, single green fluorescent protein-based redox-sensitive (roGFP) sensors adjusted for more oxidative environments enables non-invasive measurement of the ER redox environment in living cells. In turn, Förster resonance energy transfer (FRET) sensors based on two fluorophore probes represent an alternative strategy for ratiometric signal acquisition. In previous work, we described the FRET-based redox sensor CY-RL7 with a relatively high midpoint redox potential of -143 mV, which is required for monitoring glutathione potentials in the comparatively high oxidative environment of the ER. Here, the efficacy of the CY-RL7 probe was ascertained in the cytosol and ER of live cells with fluorescence microscopy and flow cytometry. The sensor was found to be fully reduced at steady state in the cytosol and became fully oxidized in response to treatment with 1-chloro-2,4-dinitrobenzene, a depletor of reduced glutathione (GSH). In contrast, the probe was strongly oxidized (88%) upon expression in the ER of cultured cells. We also examined the responsiveness of the ER sensor to perturbations in cellular glutathione homeostasis. We observed that the reductive level of the FRET sensor was increased two-fold to about 28% in cells pretreated with N-acetylcysteine, a substrate for GSH synthesis. Finally, we evaluated the responsiveness of CY-RL7 and roGFP1-iL to various perturbations of cellular glutathione homeostasis to address the divergence in the specificity of these two probes. Together, the present data generated with genetically encoded green fluorescent protein (GFP)-based glutathione probes highlight the complexity of the ER redox environment and indicate that the ER glutathione pool may be more oxidized than is currently considered.