A Comparative Study of Real-Time RT-PCR-Based SARS-CoV-2 Detection Methods and Its Application to Human-Derived and Surface Swabbed Material.

Real-time RT-PCR remains a gold standard in the detection of various viral diseases. In the coronavirus 2019 pandemic, multiple RT-PCR-based tests were developed to screen for viral infection. As an emergency response to increasing testing demand, we established a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial, one customized, and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2-positive and 92 SARS-CoV-2-negative samples. Furthermore, economical and practical characteristics of these protocols were compared. In addition, a highly sensitive digital droplet PCR (ddPCR) method was developed, and application of RT-PCR and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/µL), high sensitivities (93.6% to 97.8%), and high specificities (98.7% to 100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity, was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.