Karl Ocius

Int J Mol Sci. 2024 Jan 12;25(2):983. doi: 10.3390/ijms25020983.

ABSTRACT

Mycobacterium tuberculosis, a major cause of mortality from a single infectious agent, possesses a remarkable mycobacterial cell envelope. Penicillin-Binding Proteins (PBPs) are a family of bacterial enzymes involved in the biosynthesis of peptidoglycan. PBP4 (DacB) from M. tuberculosis (MtbPBP4) has been known to function as a carboxypeptidase, and the role and significance of carboxypeptidases as targets for anti-tuberculosis drugs or antibiotics have been extensively investigated over the past decade. However, their precise involvement remains incompletely understood. In this study, we employed predictive modeling and analyzed the three-dimensional structure of MtbPBP4. Interestingly, MtbPBP4 displayed a distinct domain structure compared to its homologs. Docking studies with meropenem verified the presence of active site residues conserved in PBPs. These findings establish a structural foundation for comprehending the molecular function of MtbPBP4 and offer a platform for the exploration of novel antibiotics.

PMID:38256057 | PMC:PMC10815838 | DOI:10.3390/ijms25020983

bioRxiv. 2023 Dec 21:2023.12.20.572362. doi: 10.1101/2023.12.20.572362. Preprint.

ABSTRACT

NOD2 is an intracellular innate immune receptor that senses bacterial peptidoglycans. Although soluble in the cytosol, a portion of the protein is associated with the plasma membrane and endosomal compartments for microbial surveillance. Palmitoylation of NOD2 by zDHHC5 promotes its membrane recruitment to drive proinflammatory and antimicrobial responses to pathogenic invasion. A depalmitoylation step by an unknown protein, thioesterase, releases NOD2 from membranes into the cytosol, where the protein can then enter a new cycle of palmitoylation-depalmitoylation. Here, we identify α/β -hydrolase domain-containing protein 17 isoforms (ABHD17A, 17B, 17C) as the thioesterases responsible for depalmitoylation of NOD2. Inhibiting ABHD17 increased the plasmalemmal localization of both wild-type NOD2 and a subset of hypo-palmitoylated Crohn's disease-associated variants, resulting in increased NF-κB activation and production of pro-inflammatory cytokines in epithelial cells. These results suggest that targeted inhibition of ABHD17 may rescue some Crohn's disease-associated NOD2 variants.

PMID:38187608 | PMC:PMC10769251 | DOI:10.1101/2023.12.20.572362

Int J Mol Sci. 2023 Dec 19;25(1):42. doi: 10.3390/ijms25010042.

ABSTRACT

Periodontitis is an oral infectious disease caused by various pathogenic bacteria, such as Porphyromonas gingivalis. Although probiotics and their cellular components have demonstrated positive effects on periodontitis, the beneficial impact of peptidoglycan (PGN) from probiotic Lactobacillus remains unclear. Therefore, our study sought to investigate the inhibitory effect of PGN isolated from L. reuteri (LrPGN) on P. gingivalis-induced inflammatory responses. Pretreatment with LrPGN significantly inhibited the production of interleukin (IL)-1β, IL-6, and CCL20 in RAW 264.7 cells induced by P. gingivalis lipopolysaccharide (LPS). LrPGN reduced the phosphorylation of PI3K/Akt and MAPKs, as well as NF-κB activation, which were induced by P. gingivalis LPS. Furthermore, LrPGN dose-dependently reduced the expression of Toll-like receptor 4 (TLR4), indicating that LrPGN inhibits periodontal inflammation by regulating cellular signaling cascades through TLR4 suppression. Notably, LrPGN exhibited stronger inhibition of P. gingivalis LPS-induced production of inflammatory mediators compared to insoluble LrPGN and proteinase K-treated LrPGN. Moreover, MDP, a minimal bioactive PGN motif, also dose-dependently inhibited P. gingivalis LPS-induced inflammatory mediators, suggesting that MDP-like molecules present in the LrPGN structure may play a crucial role in the inhibition of inflammatory responses. Collectively, these findings suggest that LrPGN can mitigate periodontal inflammation and could be a useful agent for the prevention and treatment of periodontitis.

PMID:38203215 | PMC:PMC10779245 | DOI:10.3390/ijms25010042

Proc Natl Acad Sci U S A. 2023 Dec 26;120(52):e2306863120. doi: 10.1073/pnas.2306863120. Epub 2023 Dec 21.

ABSTRACT

The gut microbiota is a considerable source of biologically active compounds that can promote intestinal homeostasis and improve immune responses. Here, we used large expression libraries of cloned metagenomic DNA to identify compounds able to sustain an anti-inflammatory reaction on host cells. Starting with a screen for NF-κB activation, we have identified overlapping clones harbouring a heterodimeric ATP-binding cassette (ABC)-transporter from a Firmicutes. Extensive purification of the clone's supernatant demonstrates that the ABC-transporter allows for the efficient extracellular accumulation of three muropeptide precursor, with anti-inflammatory properties. They induce IL-10 secretion from human monocyte-derived dendritic cells and proved effective in reducing AIEC LF82 epithelial damage and IL-8 secretion in human intestinal resections. In addition, treatment with supernatants containing the muropeptide precursor reduces body weight loss and improves histological parameters in Dextran Sulfate Sodium (DSS)-treated mice. Until now, the source of peptidoglycan fragments was shown to come from the natural turnover of the peptidoglycan layer by endogenous peptidoglycan hydrolases. This is a report showing an ABC-transporter as a natural source of secreted muropeptide precursor and as an indirect player in epithelial barrier strengthening. The mechanism described here might represent an important component of the host immune homeostasis.

PMID:38127978 | PMC:PMC10756304 | DOI:10.1073/pnas.2306863120

Obes Surg. 2023 Dec 16. doi: 10.1007/s11695-023-06989-2. Online ahead of print.

ABSTRACT

PURPOSE: Recently, the link between gut microbiota, liver inflammation, and obesity has become an interesting focus of research. The aim of this study is to show the possible relation between gut microbiota dysbiosis in patients with obesity and the presence of bacterial genomes in their liver biopsies.

MATERIALS AND METHODS: A prospective study on patients undergoing bariatric surgery was carried out. Anthropometric and metabolic data, comorbidities, stool samples, and hepatic biopsies were collected and analyzed at the time of surgery. The V3-16S rRNA region was sequenced using the Ion Torrent new-generation sequencing platform.

RESULTS: In each of the 23 patients enrolled, the bacterial population was analyzed both in the stools and liver. In eight patients (34.7%), Prevotella (62.5%), Bacteroides (50%), Streptococcus (12.5%), and Dalister (12.5%) were found in both samples, simultaneously; in 15 cases, the liver was free from colonization. The statistically significant difference between groups was a Roseburia intestinalis reduction in fecal samples of patients with liver biopsies colonized by bacteria (1% vs 3%; p = 0.0339).

CONCLUSION: To the best of our knowledge, this is the first study reporting the presence of bacterial genome in a liver biopsy on bariatric patients, instead of the microbe-associated molecular patterns. Notably, in literature, the presence of Roseburia intestinalis in stool samples has been shown to prevent intestinal inflammation playing its role in the gut barrier integrity. In our population, the Roseburia reduction was associated with the presence of bacterial genome in the liver, probably related to a greater permeability of the gut and vascular barriers.

PMID:38102371 | DOI:10.1007/s11695-023-06989-2

RSC Chem Biol. 2023 Oct 23;4(12):1014-1036. doi: 10.1039/d3cb00096f. eCollection 2023 Nov 29.

ABSTRACT

Pattern recognition receptors (PRRs) represent a re-emerging class of therapeutic targets for vaccine adjuvants, inflammatory diseases and cancer. In this review article, we summarize exciting developments in discovery and characterization of small molecule PRR modulators, focusing on Toll-like receptors (TLRs), NOD-like receptors (NLRs) and the cGAS-STING pathway. We also highlight PRRs that are currently lacking small molecule modulators and opportunities for chemical biology and therapeutic discovery.

PMID:38033733 | PMC:PMC10685800 | DOI:10.1039/d3cb00096f

J Biol Chem. 2024 Jan;300(1):105494. doi: 10.1016/j.jbc.2023.105494. Epub 2023 Nov 23.

ABSTRACT

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

PMID:38006948 | PMC:PMC10727944 | DOI:10.1016/j.jbc.2023.105494

ACS Omega. 2023 Nov 20;8(48):46073-46090. doi: 10.1021/acsomega.3c07083. eCollection 2023 Dec 5.

ABSTRACT

Bicyclo[6.1.0]nonyne (BCN) is one of the most commonly used cycloalkynes in strain-promoted azide-alkyne cycloaddition (SPAAC). The synthesis of BCN produces two diastereomers, exo-BCN and endo-BCN. The potential significance of the different steric structures of the tricyclic fused rings in SPAAC products synthesized from the BCN diastereomers has not been previously studied. We first demonstrated that only endo-BCN could reduce the level of fluorescence quenching in SPAAC reaction products. The reduction was likely due to the presence of extended tricyclic fused ring systems. This hypothesis was supported by the synthesis of a fluorescence always-on construct by substituting endo-BCN for exo-BCN in a previously reported chemical probe that was characterized with good contact fluorescence quenching. We also synthesized bis-BCN derivatives to enhance the steric structural differences in the corresponding SPAAC products. A constitutional isomer of the azido-derivatized 5(6)-carboxyfluorescein [5(6)-FAM] was reacted with both bis-exo-BCN and bis-endo-BCN compounds. However, one form of the bis-exo-BCN-based product did not augment contact fluorescence quenching, while a second bis-exo-BCN product could not further reduce contact fluorescence quenching. Nevertheless, a new fluorescence turn-on chemical probe was employed to determine the activities of two serum biomarkers, butyrylcholinesterase and paraoxonase 1. Moreover, bis-endo-BCN was exploited to successfully conjugate BSA with a 5-FAM derivative compound.

PMID:38075741 | PMC:PMC10702302 | DOI:10.1021/acsomega.3c07083

Nat Commun. 2023 Dec 2;14(1):7975. doi: 10.1038/s41467-023-43595-3.

ABSTRACT

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

PMID:38042849 | PMC:PMC10693644 | DOI:10.1038/s41467-023-43595-3

Nat Commun. 2023 Dec 4;14(1):7999. doi: 10.1038/s41467-023-43770-6.

ABSTRACT

The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. In mycobacteria and Gram-positive bacteria, the FtsEX system directly activates peptidoglycan-hydrolases by a mechanism that remains unclear. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our findings indicate that RipC is recognized through a "Match and Fit" mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.

PMID:38044344 | PMC:PMC10694151 | DOI:10.1038/s41467-023-43770-6

J Biol Chem. 2023 Dec 1:105529. doi: 10.1016/j.jbc.2023.105529. Online ahead of print.

ABSTRACT

Clostridioides difficile is the leading cause of antibiotic-associated diarrhoea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerisation of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalysed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (Ldt_Cd1, Ldt_Cd2 and Ldt_Cd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyse the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic (DAP) acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalysed by as yet unidentified non-canonical Ldt enzymes.

PMID:38043796 | DOI:10.1016/j.jbc.2023.105529

Biochem Biophys Rep. 2023 Oct 27;36:101561. doi: 10.1016/j.bbrep.2023.101561. eCollection 2023 Dec.

ABSTRACT

Sebocytes express Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which participate in the innate immune response of the skin. Although the roles of TLRs and NLR family pyrin domain-containing 3 (NLRP3) in inflammatory responses in sebocytes have been reported, the expression and functions of other NLR members, such as NOD protein-1 and -2 (NOD1 and NOD2, respectively), remain unclear. In this study, we showed that, in sebocytes, the expression of NOD1 is higher than that of NOD2, and that NOD1 is involved in inflammatory responses, such as the secretion of proinflammatory cytokines. A NOD1 agonist, L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid (Tri-DAP) induced the expression and secretion of interleukin-8 (IL-8) and activated the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways. On the other hand, a NOD2 agonist, muramyl dipeptide, did not. Either inhibition with a NOD1 inhibitor, ML130, or knockdown of NOD1 expression abolished Tri-DAP-induced inflammatory responses, suggesting that NOD1 is involved in the immunogenic signaling system of sebocytes. Furthermore, Tri-DAP and an agonist of TLR2 or TLR4 additively increased IL-8 expression compared with each agonist alone. Our results reveal the role of NOD1 in the inflammatory responses of sebocytes and may provide a novel therapeutic target for sebaceous gland inflammatory diseases, such as acne vulgaris.

PMID:37942338 | PMC:PMC10630598 | DOI:10.1016/j.bbrep.2023.101561

J Biol Chem. 2023 Nov 23:105494. doi: 10.1016/j.jbc.2023.105494. Online ahead of print.

ABSTRACT

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position 4 of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software which can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified two G. oxydans mutants from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

PMID:38006948 | DOI:10.1016/j.jbc.2023.105494

bioRxiv. 2023 Nov 21:2023.11.19.567738. doi: 10.1101/2023.11.19.567738. Preprint.

ABSTRACT

Enterococcus faecium is a microbiota species in humans that can modulate host immunity ¹ , but has also acquired antibiotic resistance and is a major cause of hospital-associated infections ² . Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity ^3-5 and immune checkpoint inhibitor antitumor activity ⁶ . However, the essential functions of SagA in E. faecium were unknown. Here we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, Δ sagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, plasmid-based expression of SagA, but not its catalytically-inactive mutant, restored Δ sagA growth, production of active muropeptides and NOD2 activation. SagA is therefore essential for E. faecium growth, stress resistance and activation of host immunity.

PMID:38014356 | PMC:PMC10680833 | DOI:10.1101/2023.11.19.567738

Bioorg Chem. 2024 Jan;142:106960. doi: 10.1016/j.bioorg.2023.106960. Epub 2023 Nov 4.

ABSTRACT

Tuberculosis is one of the major causes of death worldwide; more than a million people die every year because of this infection. The constant emergency of Mycobacterium tuberculosis resistant strains against the most used treatments also contributes to the burden caused by this disease. Consequently, the development of new alternative therapies against this disease is constantly required. In recent years, only a few molecules have reached the market as new antituberculosis agents. The mycobacterial cell wall biosynthesis is for a longstanding considered an important target for drug development. Particularly, in M. tuberculosis, the peptidoglycan cross-links are predominantly formed by nonclassical bridges between the third residues of adjacent tetrapeptides. The responsible enzymes for these reactions are ld-transpeptidases (Ldts), for which M. tuberculosis has five paralogues. Although these enzymes are distinct from the penicillin-binding proteins (PBPs), they can also be inactivated by β-lactam antibiotics, but since M. tuberculosis has a chromosomal β-lactamase, most of the antibiotics of these classes can be degraded. Thus, to identify alternative scaffolds for the development of new antimicrobials against tuberculosis, we have integrated several fragment-based drug discovery techniques. Based on that, we identified and validated a number of small molecules that could be the starting point in the synthesis of more potent inhibitors against at least two Ldts from M. tuberculosis, Ldt_Mt2 and Ldt_Mt3. Eight identified molecules inhibited the Ldts activity in at least 20%, and three of them have antimycobacterial activity. The cell ultrastructural analysis suggested that one of the best compounds induced severe effects on the septum and cell wall morphologies, which corroborates our target-based approach to identifying new Ldts hits.

PMID:37944368 | DOI:10.1016/j.bioorg.2023.106960

RSC Med Chem. 2023 Aug 30;14(11):2125-2154. doi: 10.1039/d3md00143a. eCollection 2023 Nov 15.

ABSTRACT

Antimicrobial resistance (AMR) in bacterial pathogens is a worldwide health issue. The innovation gap in discovering new antibiotics has remained a significant hurdle in combating the AMR problem. Currently, antibiotics target various vital components of the bacterial cell envelope, nucleic acid and protein biosynthesis machinery and metabolic pathways essential for bacterial survival. The critical role of the bacterial cell envelope in cell morphogenesis and integrity makes it an attractive drug target. While a significant number of in-clinic antibiotics target peptidoglycan biosynthesis, several components of the bacterial cell envelope have been overlooked. This review focuses on various antibacterial targets in the bacterial cell wall and the strategies employed to find their novel inhibitors. This review will further elaborate on combining forward and reverse chemical genetic approaches to discover antibacterials that target the bacterial cell envelope.

PMID:37974958 | PMC:PMC10650376 | DOI:10.1039/d3md00143a

Nat Immunol. 2023 Nov 13. doi: 10.1038/s41590-023-01668-x. Online ahead of print.

ABSTRACT

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

PMID:37957354 | DOI:10.1038/s41590-023-01668-x

ACS Infect Dis. 2023 Nov 9. doi: 10.1021/acsinfecdis.3c00436. Online ahead of print.

ABSTRACT

Tuberculosis (TB) is a global health threat that causes significant mortality. This review explores chemotherapeutics that target essential processes in Mycobacterium tuberculosis, such as DNA replication, protein synthesis, cell wall formation, energy metabolism, and proteolysis. We emphasize the need for new drugs to treat drug-resistant strains and shorten the treatment duration. Emerging targets and promising inhibitors were identified by examining the intricate biology of TB. This review provides an overview of recent developments in the search for anti-TB drugs with a focus on newly validated targets and inhibitors. We aimed to contribute to efforts to combat TB and improve therapeutic outcomes.

PMID:37944023 | DOI:10.1021/acsinfecdis.3c00436

Methods Mol Biol. 2024;2715:197-205. doi: 10.1007/978-1-0716-3445-5_12.

ABSTRACT

Most bacterial secretion systems are large machines that cross the cell envelope to deliver effectors outside the cell or directly into target cells. The peptidoglycan layer can therefore represent a physical barrier for the assembly of these large machines. Secretion systems and their counterparts such as type IV pili, flagella, and conjugation machines have therefore evolved or hijacked enzymes with peptidoglycan degradation activity. These enzymes are usually glycoside hydrolases that cleave the glycan chains of the peptidoglycan. Their activities are spatially controlled to avoid cell lysis and to create local rearrangement of the cell wall. In addition, peptidoglycan hydrolases may not be only required for the proper assembly of the secretion systems but may directly participate to the release of the effectors. Finally, several antibacterial effectors possess peptidoglycan degradation activity that damage the cell wall once delivered in the target cell. Here, we describe protocols to test the peptidoglycan degradation activity of these proteins in vitro and in solution.

PMID:37930529 | DOI:10.1007/978-1-0716-3445-5_12