Rapid High-Yield Production of Functional SARS-CoV-2 Receptor Binding Domain by Viral and Non-Viral Transient Expression for Pre-Clinical Evaluation.

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.

Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury.

Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.

Validation of glaucoma-like features in the rat episcleral vein cauterization model.

BACKGROUND: Glaucoma, an irreversible optic nerve neuropathy, always results in blindness. This study aimed to evaluate glaucoma-like features in the rat episcleral vein cauterization (EVC) model by multiple in vivo and in vitro evidences. METHODS: Wistar rat was used in this study. The elevated intraocular pressure (IOP) was induced by cauterization of three episcleral veins. IOP was monitored with Tono-Pen XL tonometer. Time-dependent changes to the neuronal retinal layers were quantified by Fourier domain-optical coherence tomography. The function of retina was evaluated by electroretinogram (ERG). Survival of retinal ganglion cells (RGCs) was quantified by retrograde labeling. Histology study was performed with retinal sections stained with hematoxylin-eosin, glial fibrillary acidic protein, and neuronal nuclear antigen. Retina and aqueous humor protein were extracted and cytotoxic protein tumor necrosis factor alpha (TNF-α) and alpha-2 macroglobulin (α2m) were measured with Western blotting. RESULTS: EVC is a relatively facile intervention, with low failure rates (<5%). After surgical intervention, chronic mild IOP elevation (about 1.6-fold over normal, P < 0.05) was induced for at least 6 weeks without requiring a second intervention. High IOP causes chronic and progressive loss of RGCs (averaging about 4% per week), progressive thinning of neuronal retinal layers (3-5 µm per week), and reduction of a- and b-wave in ERG. EVC method can also induce glial cell activation and alterations of inflammation proteins, such as TNF-α and α2m. CONCLUSION: EVC method can establish a robust, reliable, economic and highly reproducible glaucomatous animal model.

The influence of tumor-host interactions in the stromal cell-derived factor-1/CXCR4 ligand/receptor axis in determining metastatic risk in breast cancer.

The chemokine stromal cell-derived factor-1 (SDF-1) may function to attract CXCR4-expressing cancer cells to metastatic organs. We have previously demonstrated that low plasma SDF-1, a host-derived marker, increases distant metastatic risk in breast cancer. We therefore hypothesized that tumors overexpressing the SDF-1 receptor CXCR4 have an enhanced ability to metastasize in patients with low plasma SDF-1 levels. In this study, we determined the prognostic significance of activated CXCR4, or phosphorylated CXCR4 (p-CXCR4), and CXCR7, another receptor for SDF-1. Immunohistochemistry was performed on a tissue microarray built using 237 samples from the same cohort of patients for which we measured plasma SDF-1 levels. We found that the prognostic value of p-CXCR4 expression (hazard ratio or HR, 3.95; P = 0.004) was superior to total CXCR4 expression (HR, 3.20; P = 0.03). The rate of breast cancer-specific mortality was much higher in patients with both high p-CXCR4 expression and low plasma SDF-1 levels (HR, 5.96; P < 0.001) than either low plasma SDF-1 (HR, 3.59; P = 0.01) or high p-CXCR4 expression (HR, 3.83; P = 0.005) alone. The added prognostic value of low plasma SDF-1 was only effective in patients with high p-CXCR4 expression, and as such, provides clinical validation for modulation of the metastatic potential of tumor cells by an inherent host-derived metastatic risk factor.

Verification of cell irradiation dose deposition using a radiochromic film.

We describe a technique for the MTT assay that irradiates all cells at once by a combination of couch movement and a step-and-shoot irradiation technique on a linear accelerator with 6 MV and 18 MV photon beams. In two experimental setups, we obtained maximum to minimum dose ranges of 10 for the constant MU/bin (monitor units per bin) setup and 20 for the variable MU/bin technique. The irradiation technique described is dose rate independent and it can be used on any teletherapy irradiation machine. We also employed radiochromic film dosimetry to verify dose delivered in each of the wells within the dish. It is shown that for the lowest doses, relative dose variation within wells reaches a value of 6%. We also demonstrated that the radiochromic film positioned below the 96-well plate does not underestimate dose deposited within each compartment by more than 2% due to the vertical dose gradient.

Functional consequences of the in-frame insertion of a transposon into the mutated gamma amino butyric acid transporter of Saccharomyces cerevisiae.

BACKGROUND: The Saccharomyces cerevisiae strain, 22574d, lacks gamma amino butyric acid (GABA) transport activity and cannot grow on this amino acid as sole nitrogen source. Both transport of and growth on this amino acid are restored when the yeast is transformed with a form of mouse gamma actin containing an extended C-terminal sequence (M-g-A). The nature of the mutation in the transporter as well as the complementation mode are addressed. MATERIAL/METHODS: The cDNA sequence of the mutated transporter was achieved using reverse transcription, 3'-Race and cloning. For detection, Northern blotting and labeling with 32-P were used. The putative ability of the transporter to interact with gamma actin in vivo was examined by following the interaction in vitro of synthetic peptides corresponding to the C-termini of the gamma actin and the transporter. RESULTS: Up to codon 394 the mutated and native transporters are identical. At the 3' end, the mutant is by extended by 32 codons from the delta region of a Ty1 transposon, giving an open reading frame of 426 codons, and a predicted structure of 9 of the 11 transmembrane domains. Peptides corresponding to the carboxy terminal regions of the truncated transporter and the elongated species of gamma actin show the potential to interact in vitro. CONCLUSIONS: The mutated GABA transporter mRNA contains an insert from the delta region of a Ty1 transposon. This insertion allows expression of a transporter capable of interacting with elongated gamma actin to rehabilitate the transporter.

New templates for syntheses of ring-fused, C(10) beta-turn peptidomimetics leading to the first reported small-molecule mimic of neurotrophin-3.

beta-Turn peptidomimetics 1 were designed to mimic hot spots of neurotrophin-3 (NT-3) and others. Solid-phase syntheses of these were developed, though limitations were encountered with scale-up. Consequently, an alternative design with 2 was investigated. 1 and 2 favored distorted type I beta-turn conformations in solution. It was found that peptidomimetic 2b has NT-3-like neurotrophic activity in cell survival assays, selectively binds the NT-3 receptor TrkC, and induces the tyrosine phosphorylation of the TrkC receptor.