Dsouza, A. | bioRxiv Science

bioRxiv2021

A large-volume sputum dry storage and transportation device for molecular and culture-based diagnosis of tuberculosis

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in native Standard 17 glass fiber was stable for molecular testing after 10-day storage at 45{degrees}C, and for culture testing after 10- and 5-day storage at 37{degrees}C and 45{degrees}C, respectively. Compatibility with human sputum storage was demonstrated by dry storing Mycobacterium bovis-spiked pooled human sputum in SPECTRA-tube for 5 days at room temperature followed by successful qPCR detection. By significantly increasing the volume of samples that can be transported in the dry state and enabling recovery of the entire sample in liquid state, SPECTRA-tube presents a potential universal solution for the preservation and transportation of liquid specimens.

bioengineering↗

bioRxiv2020

Wnt-inducible Lrp6-APEX2 Interacting Proteins identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as {beta}-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of {beta}-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/{beta}-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling. SignificanceWnt/{beta}-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylated-enriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling.

cell biology↗