Biology subjects

Segatori, L.

Publications and source records attributed to Segatori, L..

3 recordsLinked to original sources

bioRxiv2025

Establishing an RNA sensor with high sensitivity and dynamic range utilizing a signal amplifier platform

Precise control of gene expression in a cell-state-specific manner is essential for effective therapeutic interventions in complex and dynamic disease microenvironments. Traditional targeting strategies that rely on surface markers or cell type-specific promoters often assume static cellular identities, limiting effectiveness in contexts such as cancer and inflammation, where cell states are highly heterogeneous and dynamic. RNA sensors, such as RADAR (RNA sensing using Adenosine Deaminases Acting on RNA), provide a modular, programmable, and non-integrating platform for classifying cell states. However, it is also characterized by low sensitivity and dynamic range, which limits its applications in detecting low-abundance transcripts. In this work, we integrate RADAR sensors with a signal amplification circuit to enhance sensitivity and dynamic range. We demonstrate that this combined RADAR-amplifier platform enables real-time monitoring of subtle changes in the abundance of endogenous transcripts under physiological conditions. Our results demonstrate the utility of this platform for fundamental biological studies and the development of precision therapeutic strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/675666v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10a05baorg.highwire.dtl.DTLVardef@fe118aorg.highwire.dtl.DTLVardef@318dfforg.highwire.dtl.DTLVardef@16172e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Localized delivery of corticosteroids via in situ modification of gut commensals using a synthetic stem peptide prodrug

Oral colonic drug delivery systems (CDDSs) are oftentimes associated with a short duration of action and poor tissue specificity. To address these challenges, we engineered an oral prodrug that leverages the engraftment and semi-permanence of gut commensals to create a long-acting colonic drug depot. We show that two synthetic stem peptide probes can be stereoselectively incorporated onto the surface of gut bacteria in C57BL/6 mice following oral administration. We then show that a prodrug consisting of budesonide, a corticosteroid with otherwise limiting side effects used to treat ulcerative colitis (UC), conjugated to one of these probes via a hydrolyzable ester is significantly less bioactive and is cleaved over a period of days to weeks in simulated physiological fluids. This prodrug can be integrated into the bacterial peptidoglycan in vitro and be cleaved into free budesonide over time, thereby improving drug localization and potentially rendering it safer for longer-term use. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/632432v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@2fa797org.highwire.dtl.DTLVardef@a3b5e2org.highwire.dtl.DTLVardef@1fa11fdorg.highwire.dtl.DTLVardef@b316c7_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCECorticosteroids are highly effective anti-inflammatory drugs used in the treatment of a variety of conditions. Unfortunately, long-term corticosteroid ingestion can lead to a host of dangerous and undesirable side effects including osteoporosis, glaucoma, and a higher risk of infection, among others. Topical corticosteroids delivered via inhalation (chiefly, budesonide and fluticasone) are the primary long-term treatment modality for chronic asthma symptoms. In contrast to oral corticosteroids, they are considered safer for long-term use when given in moderation because they are directly applied to the airways and exhibit low systemic bioavailability. We sought to apply this successful paradigm to another autoimmune-related disease, ulcerative colitis (UC). We developed a drug delivery system that combines the weakly targeting method of ingestion with a highly specific parameter, microbe prevalence along the gastrointestinal tract, to help improve the specificity and colonic retention of the corticosteroid budesonide, which is currently limited to being used as a short-term treatment for moderate-to-severe UC. Our approach utilizes a largely inert prodrug that can be incorporated into the peptidoglycan of commensal bacteria found at high densities in the colon. After tethering to the bacterial surface via a synthetic stem peptide, the prodrug passively hydrolyzes (cleaves) to release the active, unadulterated form of the drug into the local area, whereas prodrug that traffics elsewhere has a higher chance of being cleared from the body before cleavage. In this manner, we can achieve targeted immunosuppression and sustained release, rendering corticosteroids, and potentially other small molecules, safer for longer term use in treating patients with UC.

bioengineering↗

bioRxiv2024

Novel approaches to label the surface of S. aureus with DBCO for click chemistry-mediated deposition of sensitive cargo

The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications, including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for in situ conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity for some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of Staphylococcus aureus and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes - a sortase peptide substrate (LPETG), two D-alanine derivatives, and vancomycin - can selectively label the S. aureus surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-D-alanine variants do not adversely affect the growth of S. aureus or lead to off-target labeling or toxicity in HEK293T cells, even at high concentrations. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified S. aureus. We believe the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of S. aureus and other gram-positive bacteria, opening the door for new applications using azido-modified cargo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/629304v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16c7d1corg.highwire.dtl.DTLVardef@189bd12org.highwire.dtl.DTLVardef@1dc1bcborg.highwire.dtl.DTLVardef@1a4e1b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗