GDNF protein, human

C57BL6 timed pregnant mice (Charles Rivers) were sacrificed following the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. Primary motor neurons from E13.5 mouse embryos were isolated and cultured essentially as described [30 (link),31 (link)], but the magnetic column step was omitted, and 6% Optiprep (Sigma) was used for gradient centrifugation. Glia cells obtained from embryonal spinal cords after motor neuron isolation were grown in MEM, 10%FBS, 10 mM Hepes, 33 mM glucose. Secondary glia cells were incubated with motor neuron culture medium (Neurobasal, 0.5 mM Glutamax, 2% B27; Invitrogen) for one day to obtain glia-conditioned media. Motor neurons were plated on 15 mm coverslips (Assistant cover glasses; Carolina) coated with 0.5 mg/ml poly-ornithine (MW 30-70 KDa; Sigma) and 0.5 mg/ml laminin (Invitrogen), and cultured in glia-conditioned Neurobasal/B27 medium supplemented with 2% horse serum (Sigma), and 10 ng/ml each BDNF, CNTF, and GDNF (Peprotech).

The dataset published by Koch et al. [4 (link)] was used as a starting point for the Sensbio database. It contains a 2018 collection of TF-ligand interactions from different databases and literary resources. To expand and update this dataset, data dumps detailing aTFs and their triggering compounds were collected, cleaned and formatted accordingly from the following databases: BioNemo [5 (link)], RegulonDB [6 (link)], RegPrecise [7 (link)], RegTransBase [8 (link)], Sigmol [9 (link)] and GroovDB [10 (link)].
Custom Python 3 scripts (using standard libraries like Pandas and Numpy) were used to populate, clean, format and analyze the database and to build a web application through the Streamlit framework (https://streamlit.io/). Molecular fingerprints were extracted, analyzed and compared using the RDKit python library [11 ]. Networkx python module was used to describe and produce the molecular network. A local BLAST+ installation allowed the scoring and ranking of the protein sequences. Ete3 python toolkit [12 (link)] produced the phylogenetic trees of the TF sequences. Deep learning techniques were applied to build the predictive model through the Tensorflow and Keras Python libraries.
Classyfire [13 (link)] and iFragment [14 (link)] external web applications were used to classify the different molecules by chemical and metabolic categories respectively. Classyfire produces a hierarchical list of ontologies. In this case, the parent ontology was kept as the representative category for each molecule. iFragment on the other hand, produces a list of KEGG [15 (link)] metabolic pathways ordered by the probability of the input compound to belong to that particular pathway. The three pathways with the lowest p-value were selected. Using the KEGG restful API (https://www.kegg.jp/kegg/rest/keggapi.html), the parent ontology was extracted for each pathway and assigned as the final metabolic category.

All animal preparations adhered to protocols approved by the Institutional Animal Care and Use Committee of the Veterans Administration Connecticut Healthcare System. DRG neurons were isolated from neonatal Sprague–Dawley rats and transfected with HaloTag-Na_V1.8 as previously described (Dib-Hajj et al., 2009 (link); Higerd-Rusli et al., 2022 (link)). Studies of channel traffic used two microfluidic chambers separated by a physical barrier 450 μm in width with microgrooves to allow axons to extend from a soma chamber to the axon chamber (Xona Microfluidics, Research Triangle Park, NC) that were adhered to 50 mm glass-bottom dishes previously coated with poly-L-lysine and laminin as described (Akin et al., 2021 (link)). Transfected DRG neurons were applied to the somatic chambers and the growth medium (Neurobasal with 2% B27, 1% penicillin/streptomycin, 1% GlutaMAX, all from Thermo Fisher Scientific, Waltham, MA) was supplemented with 50 ng/ml NGF (Envigo, Indianapolis, IN) and GDNF (Preprotech, Windsor, NJ) the following day. The distal axonal chamber received medium with 100 ng/ml NGF and GDNF to attract growth of axons into this chamber. Cultures were treated as described below and analyzed on day 7 after preparation unless otherwise noted.