Covering: up to the end of 2020
Nonribosomal peptide synthetases are remarkable molecular machines that produce a wide range of structurally complex peptide natural products with important ...applications in medicine and agriculture. Condensation domains play a central role in these biosynthetic pathways by catalysing amide bond formation between various aminoacyl substrates. In recent years, however, it has become increasingly clear that the catalytic repertoire of C domains extends far beyond conventional peptide bond formation. C domains have been shown to perform highly diverse functions during nonribosomal peptide assembly, such as β-lactam formation, dehydration, hydrolysis, chain length control, cycloaddition, Pictet-Spengler cyclization, Dieckmann condensation and recruitment of auxiliary enzymes. In this review, a comprehensive overview of the multifaceted role of C domains in the biosynthesis of specialized metabolites in bacteria and fungi is presented. Different perspectives are also offered on how the exceptional functional versatility of C domains may be exploited for bioengineering approaches to expand the chemical diversity of nonribosomal peptides and other natural products.
Condensation domains perform highly diverse functions during natural product biosynthesis and are capable of generating remarkable chemical diversity.
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is ...responsible for the biosynthesis of the oximidine anticancer agents, oxime‐substituted benzolactone enamides that inhibit vacuolar H+‐ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O‐methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans‐AT PKSs and identify potential strategies for the production of novel oximidine analogues.
The anticancer agent oximidine I and three novel variants were discovered as products of a cryptic trans‐AT PKS/NRPS in Pseudomonas baetica. By manipulating the biosynthetic pathway, a key intermediate was identified that retains potent anticancer properties. A combination of bioinformatics analysis and genetic and biochemical experiments illuminated the oximidine biosynthetic pathway, including a novel mechanism for O‐methyloxime formation.
Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational ...change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.
•Anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation.•An open ADAMTS13 conformation is a novel and sensitive biomarker for subclinical iTTP.
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Purpose
To evaluate diagnostic accuracy, safety, and efficiency of an MRI-TRUS fusion-guided transperineal prostate biopsy method in an outpatient setting under local anaesthesia.
Methods
Patients ...undergoing transperineal prostate biopsy were included from March 2021 to May 2022. Biopsies were performed under local anaesthesia in an outpatient setting, using specialised fusion software. Primary outcome was (clinically significant) cancer detection rate. Secondary outcomes were procedure time, patient discomfort during the procedure and complication rate.
Results
We included 203 male patients (69 years +−SD 8.2) with PI-RADS score > 2. In total 223 suspicious lesions were targeted. Overall cancer detection rate and clinically significant cancer detection rate were 73.5% and 60.1%, respectively. (Clinically significant) cancer detection rates in PI-RADS 3, 4 and 5 lesions were 46.4% (23.2%), 78.5% (66.1%) and 93.5% (89.1%), respectively. Mean duration of the procedure including fusion, targeted and systematic biopsies was 22.5 min. Patients rated injection of local anaesthesia on a numeric pain rating scale on average 3.7/10 (SD 2.09) and biopsy core sampling 1.6/10 (SD 1.65). No patient presented with acute urinary retention on follow-up consultation. Two (1%) patients presented with infectious complications. Four (2%) patients experienced a vasovagal reaction.
Conclusion
Transperineal targeted biopsy with MRI-TRUS fusion software has high overall and clinically significant cancer detection rates. The method is well tolerated under local anaesthesia and in an outpatient setting.
Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the ...luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.
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