Cortes-Llanos, B. | bioRxiv Science

bioRxiv2023

Impact of magnetite nanowires orientation on morphology and activity of in vitro hippocampal neural networks

Nanomaterials design, synthesis and characterization are ever-expanding approaches towards developing biodevices or neural interfaces to treat neurological diseases. The ability of nanomaterials features, to tune neuronal networks morphology or functionality is still under study. In this work, we unveil how, when interfacing mammalian brain cultured neurons, iron oxide nanowires (NWs) orientation affects neuronal and glial densities, and network activity. Iron oxide NWs were synthesized by electrodeposition, fixing the diameter to 100 nm and the length to 1 m. Scanning electron microscopy, Raman and contact angle measurements were performed to characterize the NWs morphology, chemical composition and hydrophilicity. Hippocampal cultures were seeded on NWs devices and after 14 days the cell morphology was studied by immunocytochemistry and confocal microscopy. Live calcium imaging was performed to study neuronal activity. Using random (R-NWs) a higher neuronal and glial cell densities were obtained compared with the control and vertical (V-NWs), while using V-NWs more stellate glial cells were found. R-NWs produced a reduction in neuronal activity while V-NWs increased the neuronal network activity, possibly due to higher neuronal maturity and a lower number of GABAergic neurons, respectively. These results highlight the potential of NWs manipulations to design ad hoc regenerative interfaces.

bioengineering↗

bioRxiv2023

Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation

A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.

bioengineering↗