Microneedle technologies have the potential for expanding the capabilities of wearable health monitoring from physiology to biochemistry. This paper presents the fabrication of silicon hollow microneedles by a deep-reactive ion etching (DRIE) process, with the aim of exploring the feasibility of microneedle-based in-vivo monitoring of biomarkers in skin fluid. Such devices shall have the ability to allow the sensing elements to be integrated either within the needle borehole or on the backside of the device, relying on capillary filling of the borehole with dermal interstitial fluid (ISF) for transporting clinically relevant biomarkers to the sensor sites. The modified DRIE process was utilized for the anisotropic etching of circular holes with diameters as small as 30 μm to a depth of >300 μm by enhancing ion bombardment to efficiently remove the fluorocarbon passivation polymer. Afterward, isotropic wet and/or dry etching was utilized to sharpen the needle due to faster etching at the pillar top, achieving tip radii as small as 5 μm. Such sharp microneedles have been demonstrated to be sufficiently robust to penetrate porcine skin without needing any aids such as an impact-insertion applicator, with the needles remaining mechanically intact after repetitive penetrations. The capillary filling of DRIE-etched through-wafer holes with water has also been demonstrated, showing the feasibility of use to transport the analyte to the target sites.

Pevonedistat (MLN4924), a specific NEDD8‐activating enzyme inhibitor, has been considered as a promising treatment for glioblastoma, which is currently in Phase I/II clinical trials. On the other hand, inhibition of neddylation pathway substantially upregulates the expression of T cell negative regulator programmed death‐ligand 1 (PD‐L1), which might account for the potential resistance via evasion of immune surveillance checkpoints. Whether administration of anti‐PD‐L1 enhances the efficacy of pevonedistat through a cytotoxic T cell‐dependent mechanism in glioblastoma needs to be investigated. Here, we report that depletion of neddylation pathway key enzymes markedly elevates PD‐L1 expression in glioblastoma cancer cells. Consistently, neddylation inhibitor pevonedistat significantly enhances PD‐L1 expression in both glioblastoma cancer cell lines and animal models. Mechanistically, pevonedistat increases PD‐L1 mRNA levels mainly through inhibiting Cullin1‐F‐box and WD repeat domain‐containing 7 E3 ligase activity and accumulating c‐MYC proteins, a direct transcriptional activator of PD‐L1 gene expression. In addition, inhibition of Cullin3 activity by pevonedistat also blocks PD‐L1 protein degradation. Importantly, pevonedistat attenuates T cell killing through PD‐L1 induction, and blockade of PD‐L1 restores the sensitivity of pevonedistat‐treated glioblastoma cancer cells to T cell killing. The combination of pevonedistat and anti‐PD‐L1 therapy compared to each agent alone significantly increased the therapeutic efficacy in vivo. Our study demonstrates inhibition of neddylation pathway suppresses cancer‐associated immunity and provides solid evidence to support the combination of pevonedistat and PD‐L1/programmed cell death protein 1 immune checkpoint blockade as a potential therapeutic strategy to treat glioblastoma.

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Rationale:Children usually develop less severe symptoms responding to Coronavirus Disease 2019 (COVID-19) than adults. However, little is known about the molecular alterations and pathogenesis of COVID-19 in children. Methods:We conducted plasma proteomic and metabolomic profilings of the blood samples of a cohort containing 18 COVID-19-children with mild symptoms and 12 healthy children, which were enrolled from hospital admissions and outpatients, respectively. Statistical analyses were performed to identify molecules specifically altered in COVID-19-children. We also developed a machine learning-based pipeline named inference of biomolecular combinations with minimal bias (iBM) to prioritize proteins and metabolites strongly altered in COVID-19-children, and experimentally validated the predictions. Results: By comparing to the multi-omic data in adults, we identified 44 proteins and 249 metabolites differentially altered in COVID-19-children against healthy children or COVID-19-adults. Further analyses demonstrated that both deteriorative immune response/inflammation processes and protective antioxidant or anti-inflammatory processes were markedly induced in COVID-19-children. Using iBM, we prioritized two combinations that contained 5 proteins and 5 metabolites, respectively, each exhibiting a total area under curve (AUC) value of 100% to accurately distinguish COVID-19-children from healthy children or COVID-19-adults. Further experiments validated that all the 5 proteins were up-regulated upon coronavirus infection. Interestingly, we found that the prioritized metabolites inhibited the expression of pro-inflammatory factors, and two of them, methylmalonic acid (MMA) and mannitol, also suppressed coronaviral replication, implying a protective role of these metabolites in COVID-19-children. Conclusion:The finding of a strong antagonism of deteriorative and protective effects provided new insights on the mechanism and pathogenesis of COVID-19 in children that mostly underwent mild symptoms. The identified metabolites strongly altered in COVID-19-children could serve as potential therapeutic agents of COVID-19.

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