Rachita Dash

Nat Chem Biol. 2025 Apr 28. doi: 10.1038/s41589-025-01891-7. Online ahead of print.

ABSTRACT

Monitoring H₂O₂ dynamics in conjunction with key biological interactants is critical for elucidating the physiological outcome of cellular redox regulation. Optogenetic hydrogen peroxide sensor with HaloTag with JF635 (oROS-HT₆₃₅) allows fast and sensitive chemigenetic far-red H₂O₂ imaging while overcoming drawbacks of existing red fluorescent H₂O₂ indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT₆₃₅ with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT₆₃₅ and multiplexed H₂O₂ imaging enables spatially resolved imaging of H₂O₂ targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT₆₃₅ with other green fluorescence reporters by capturing acute and real-time changes in H₂O₂ with intracellular redox potential and Ca²⁺ levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT₆₃₅ enables detailed insights into intricate intracellular and intercellular H₂O₂ dynamics, along with their interactants, through spatially resolved, far-red H₂O₂ imaging in real time.

PMID:40295764 | DOI:10.1038/s41589-025-01891-7

Org Biomol Chem. 2025 May 6. doi: 10.1039/d5ob00627a. Online ahead of print.

ABSTRACT

The relationship between intracellular uptake efficacy and the folding behavior of arginine-rich cell-penetrating L/D-peptides with α,α-disubstituted α-amino acids in plasmid DNA (pDNA) delivery was examined. Nano-sized complexes formed from pDNA and L/D-peptides efficiently traversed the cell membrane regardless of the peptide conformation. This finding represents a significant deviation from previously reported covalent cargo delivery methods using cell penetrating peptides with L- and D-amino acids.

PMID:40325951 | DOI:10.1039/d5ob00627a

Chembiochem. 2025 Apr 9:e202500032. doi: 10.1002/cbic.202500032. Online ahead of print.

ABSTRACT

Cell-penetrating peptides (CPPs) are favored for protein delivery due to their efficiency, rapidity, and low toxicity. However, conjugation of CPPs to proteins often requires significant amounts of CPPs to ensure yields, which also may result in increased proteins dimer formation. Here, we report that the use of low equivalents 2,2'-dithiodipyridine (DPDS)-activated CPP for conjugation cargo allows for high-conversion-rate CPP-cargo conjugates. Using this strategy, we obtained high-conversion-rate conjugates of cyclic deca-arginine peptide (cR10) with ubiquitin (Ub) and UbcH7 using only low equivalents of cR10. Furthermore, we successfully conjugate three CPPs to cargo via DPDS and confirmed its successful cytosolic delivery through fluorescence imaging.

PMID:40200819 | DOI:10.1002/cbic.202500032

Nat Commun. 2025 Apr 14;16(1):3535. doi: 10.1038/s41467-025-58676-8.

ABSTRACT

Bacterial outer membrane vesicles (OMVs) are nano-sized structures derived from the outer membrane of Gram-negative bacteria, which have emerged as key players in host-pathogen interactions, yet their potential as biomarkers remains largely unexplored due to the difficulty of identification in complex biological samples. Here we show an approach for detecting and quantifying bacterial OMVs in blood using a Polymyxin B-fluorescein probe (PmBF), which targets bacterial lipopolysaccharides (LPS). The probe selectively labels OMVs, enabling their differentiation from host extracellular vesicles and quantitative analysis using nano-flow cytometry. In male mouse models of pneumonia, we observe elevated serum PmBF⁺ EVs as early as 6 h post-infection, preceding positive blood cultures. In clinical samples, PmBF⁺ EVs show superior performance for diagnosing bacterial infections and differentiate them from virus or mycoplasma infections. Our findings highlight circulating PmBF⁺ EVs as promising biomarkers of bacterial infections.

PMID:40229269 | PMC:PMC11997070 | DOI:10.1038/s41467-025-58676-8

ACS Med Chem Lett. 2025 Mar 20;16(4):638-645. doi: 10.1021/acsmedchemlett.5c00037. eCollection 2025 Apr 10.

ABSTRACT

A majority of drugs are small molecules that satisfy Lipinski's Rule-of-Five (Ro5), but efforts to target topologically complex biomolecular interactions have reignited interest in nonconforming molecular therapeutics, dubbed "beyond Ro5 (bRo5)". Broadly useful design principles for bRo5 molecules are few in number, although several studies have highlighted the benefit to bioavailability and proteolytic stability that can result from the introduction of a constraining ring into conformationally mobile peptides. Here we show that a linear oligomeric depsipeptide (OD) template can be leveraged to link size to permeability, while the corresponding cyclic oligomeric depsipeptide (COD) series is used to determine the impact of cyclization as an added conformational constraint. Unexpectedly, certain macrocycle sizes confer a greater benefit to permeability than others.

PMID:40236530 | PMC:PMC11995216 | DOI:10.1021/acsmedchemlett.5c00037

iScience. 2025 Mar 16;28(4):112227. doi: 10.1016/j.isci.2025.112227. eCollection 2025 Apr 18.

ABSTRACT

The resistance of Clostridioides difficile to the β-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3→3 cross-links generated by l,d-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3→3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTs. The d,d-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the l,d-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.

PMID:40224013 | PMC:PMC11986978 | DOI:10.1016/j.isci.2025.112227

ACS Biomater Sci Eng. 2025 May 12;11(5):3003-3018. doi: 10.1021/acsbiomaterials.5c00007. Epub 2025 Apr 11.

ABSTRACT

To recapitulate prostate cancer metastasis, DU145 cells were cultured in a hyaluronic acid-based, bio-orthogonally constructed, protease-degradable hydrogels. In the presence of a covalently conjugated integrin-binding peptide (GRGDSP), DU145 cells formed tumoroids and exhibited small protrusions. Upon addition of soluble transforming growth factor beta 1 (TGFβ1), cells underwent morphological changes to form extended interconnected cellular networks. Contrarily, in RGD-free hydrogels, cells maintained spherical structures even in the presence of TGFβ1. In RGD-conjugated hydrogels, TGFβ1 induced nuclear localization of SMAD2/3, upregulating a wide range of TGFβ1 target genes and proteins. Prolonged exposure to TGFβ1 led to matrix remodeling and induced epithelial-to-mesenchymal transition in DU145 cells, with loss of epithelial markers and gain of mesenchymal markers. A pharmacological inhibitor of TGFβRI/ALK5, SB-431542, attenuated TGFβ1-induced morphological changes, abrogated nuclear localization of SMAD2/3, and restored the expression of key epithelial markers. Our findings highlight the cooperative role of TGFβ1 signaling and integrin-binding peptide in the acquisition of an aggressive phenotype and the promotion of tumor progression.

PMID:40214406 | PMC:PMC12700150 | DOI:10.1021/acsbiomaterials.5c00007

ACS Cent Sci. 2025 Mar 10;11(3):431-440. doi: 10.1021/acscentsci.4c01021. eCollection 2025 Mar 26.

ABSTRACT

Macrocyclic peptides make up a unique class of modalities known for their high affinity, specificity, and ability to modulate protein-protein interactions, including receptor activation. Messenger RNA display, including the Random Nonstandard Peptides Integrated Discovery (RaPID) system, stands out in identifying target-specific macrocyclic peptides, producing potent binders with low to subnanomolar dissociation constants against diverse targets. It has often been discussed that this success is partly attributed to the vast library of over a trillion different peptide sequences expressed from the corresponding mRNA sequences. However, the impact of library scales on the identification of various binders has not been experimentally validated. Here, we report the RaPID selections against an ectodomain of a receptor tyrosine kinase MET using peptide libraries ranging from 10⁶ to 10¹⁴ unique members of mRNAs. We thoroughly analyzed the outcomes, including the binding kinetic properties, of the enriched peptide families. This study provides valuable guidelines for designing libraries with various numbers of sequences and selection conditions to enrich macrocyclic peptides with the desired characteristics.

PMID:40161957 | PMC:PMC11950852 | DOI:10.1021/acscentsci.4c01021