Biology subjects

Santos, J. A.

Publications and source records attributed to Santos, J. A..

4 recordsLinked to original sources

bioRxiv2026

Delivery of small interfering RNA and antisense oligonucleotides across the blood-brain barrier with monovalent transferrin receptor 1 binding VHH-Fc fusion proteins

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

neuroscience↗

bioRxiv2025

The Mycobacterium tuberculosis long-chain fatty acid resistome reveals the universal stress protein TB15.3 as essential for infection

The human pathogen Mycobacterium tuberculosis (Mtb) thrives in lipid-rich microenvironments. Long-chain fatty acids (LCFA) are some of the most abundant lipids encountered by Mtb during infection. Mtb has evolved to utilize LCFA as a preferred carbon source, however, LCFA are also known to be potent antimicrobials. Mtb must therefore employ mechanisms to utilize LCFA as a carbon source while avoiding its bactericidal properties. We used transposon sequencing (TnSeq) to define a Mtb LCFA resistome, and found it to be composed by 38 genes. Surprisingly, Mtb requires a diverse set of metabolic pathways to avoid LCFA toxicity, indicating pleiotropic effects of LCFA in Mtb metabolism. As a functional follow-up on the TnSeq screen, we investigated the function of the universal stress protein TB15.3 in LCFA metabolism and during infection. We show that TB15.3 acts as a "metabolic break" for LCFA uptake and catabolism, avoiding deleterious membrane hyperpolarization. This was associated with loss of viability in the chronic phase of infection in mice and in an in vitro caseum model. Our work highlights Mtb LCFA resistance mechanisms as an important adaptation to the host and a promising target space to be exploited for drug development.

microbiology↗

bioRxiv2023

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function

The mycobacterial mutasome - minimally comprising ImuA, ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the {beta} clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA, a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

microbiology↗

bioRxiv2019

Whole integration of neural connectomics, dynamics and bio-mechanics for identification of behavioral sensorimotor pathways in Caenorhabditis elegans

Computational approaches which emulate in-vivo nervous system are needed to investigate mechanisms of the brain to orchestrate behavior. Such approaches must integrate a series of biophysical models encompassing the nervous system, muscles, biomechanics to allow observing the system in its entirety while supporting incorporations of different model variations. Here we develop modWorm: a modeling framework for the nematode Caenorhabditis elegans using modular integration approach. modWorm allows for construction of a model as an integrated series of configurable, exchangeable modules each describing specific biophysical processes across different modalities (e.g., nervous system, muscles, body). Utilizing modWorm, we propose a base neuro-mechanical model for C. elegans built upon the complete connectome. The model integrates a series of 7 modules: i) intra-cellular dynamics, ii) electrical and iii) chemical extra-cellular neural dynamics, iv) translation of neural activity to muscle calcium dynamics, v) muscle calcium dynamics to muscle forces, vi) muscle forces to body postures and vii) proprioceptive feedback. We validate the base model by in-silico injection of constant currents into sensory and inter-neurons known to be associated with locomotion behaviors and by applying external forces to the body. Applications of in-silico neural stimuli experimentally known to modulate locomotion show that the model can recapitulate natural behavioral responses such as forward and backward locomotion as well as mid-locomotion stimuli induced responses such as avoidance and turns. Furthermore, through in-silico ablation surveys, the model can infer novel neural circuits involved in sensorimotor behaviors. To further dissect mechanisms of locomotion, we utilize modWorm to introduce empirical based variations of intra and extra-cellular dynamics as well as model optimizations on associated parameters to elucidate their effects on simulated locomotion dynamics compared to experimental findings. Our results show that the proposed framework can be utilized to identify neural circuits which control, mediate and generate natural behavior.

neuroscience↗