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Spinal Muscular Atrophy (SMA) Type II is a neurodegenerative disease that leads to progressive muscle weakness. It prevents children from walking and affects their respiratory function and their activity tolerance, among other health problems. We aimed to assess the activity tolerance showed by a child with SMA using a pediatric gait exoskeleton at home when walking and performing activities.This study presents the case of a 6-year-old boy with SMA Type II and respiratory failure who used a pediatric gait exoskeleton at home for a period of two months. A nursing assessment was done before and during the use of the device to evaluate the child's activity tolerance during the sessions. Nursing interviews, performance, vital signs, fatigue, field notes, and functional scales were analyzed.The nursing assessment showed a good activity tolerance of the child. Performance using the device improved over time; vital signs did not vary significantly during the sessions; fatigue perception decreased over time; and the child reached a higher score on some functional outcomes.A first step has been taken to evaluate the impact of exoskeleton technology in children with SMA Type II from the nursing point of view, exposing the potential of this technology for the care of children with neuromuscular diseases, and the need for more research on the topic.The information in this study will be useful to nurses to know the effects of gait exoskeletons in pediatric care of children with neuromuscular diseases like SMA.

Seven new polyketides, termed veramycins, were isolated from a Streptomyces sp. from the Sanofi microbial strain collection along with their known congeners NFAT-133 and TM-123. Veramycin A, an α-pyrone congener of TM-123 and NFAT-133 showed an increased baseline deoxy-glucose uptake in the absence of insulin in a modified L6 rat skeletal muscle cell line (L6 GLUT4 AS160-like cells). In addition, both compounds slightly increased the sensitivity to insulin in this cell line. Total syntheses of NFAT-133, TM-123 and veramycin A were accomplished starting from a central building block, which bears the three contiguous stereogenic centers of this polyketide family. Our approach enables an efficient, selective and flexible access to all possible isomers of the stereotriad for further exploration of this series as a potential anti-diabetic lead structure as exemplified by the synthesis of an NFAT-133 epimer. Finally, the corresponding biosynthetic gene cluster (BGC) was identified by genome sequencing and gene inactivation. Based on feeding experiments, a biosynthetic pathway was proposed, which enabled access to new veramycin A analogs by precursor-directed biosynthesis.

BACKGROUND: Cerebral Palsy (CP) and Spinal Muscular Atrophy (SMA) are common causes of motor disability in childhood. Gait exoskeletons are currently being used as part of rehabilitation for children with walking difficulties. OBJECTIVE: To assess the safety and efficacy and describe the main characteristics of the clinical articles using robot-assisted gait training (RAGT) with exoskeleton for children with CP or SMA. METHODS: A computer search was conducted in five bibliographic databases regarding clinical studies published in the last ten years. In order to be included in this review for further analysis, the studies had to meet the following criteria: (1) assess efficacy or safety of interventions; (2) population had to be children with CP or SMA aged between 3 and 14; (3) exoskeleton must be bilateral and assist lower limbs during walking. RESULTS: Twenty-one articles were selected, of which only five were clinical trials. 108 participants met the inclusion criteria for this study, all with a diagnosis of CP. The evidence level of the selected papers was commonly low. CONCLUSIONS: RAGT therapy seems to be safe for children with CP. However, further investigation is needed to confirm the results related to efficacy. There is no evidence of RAGT therapy for SMA children.

1,2-unsaturated pyrrolizidine alkaloids are found naturally in Symphytum officinale, well known as comfrey, which has a longstanding use for the topical treatment of painful muscle and joint complaints. Pyrrolizidine alkaloids (PA) are a relevant concern for the safety assessment due to their liver genotoxicity profile, and close attention is paid during manufacturing to minimizing their levels. Current regulatory risk assessment approaches include setting limits that derive from toxicity data coming from the oral route of exposure. This study investigated to what extent pyrrolizidine alkaloids are bioavailable following topical exposure, assessing penetration of retronecine-type PAs in an in vitro human skin model. A single comfrey root formulation was spiked with 3 different congeners (a 7R-monoester, an open-chained 7R-diester, and a cyclic diester) and percutaneous absorption measured per OECD guidelines and good laboratory practices. The measured penetration for all 3 PAs was low and compared favourably with existing in vitro data. Although consideration of different regulatory guidance influences the determination of dermally absorbed dose, these data facilitate the understanding of absorption differences following topical exposure, which in turn can be taken into account in the risk assessment.

The structures of five linear lipopeptides, thaxteramides A1, A2, B1, B2, and C isolated from the myxobacterium Jahnella thaxteri, were elucidated. They have a C-terminal common tetrapeptidic Tyr-Gly-β-Ala-Tyr core but differ in the stereochemistry of the tyrosine units, methylations, the remaining amino acids, and the N-terminal polyketide. In silico analysis of the genome sequence complemented with feeding experiments revealed two distinct hybrid PKS/NRPS gene clusters. Three semisynthesized cyclic analogues were found to inhibit the growth of Gram-positive bacteria.

Medical and rehabilitation exoskeletons are being increasingly considered by therapists when choosing a treatment for individuals affected by lower limb impairments. Although all such exoskeletons seem to provide similar features and performance, there are, in practice, significant differences among them in terms of maximum walking speed, maximum torque, weight, autonomy, interaction with the user, or even the way to use it. In this review, the state of the art of the main commercial exoskeletons is described, while analyzing their properties, advantages, and disadvantages. Three groups are considered: complete exoskeletons, partial exoskeletons and open lines of research. A comparative analysis between them is performed while considering the main scientific and technical aspects to be improved. In conclusion to this analysis, the balance between feasibility and innovation in exoskeletons development is a design challenge. Commercial exoskeletons must fulfil standards whilst ensuring their safety and robustness. However, achieving a new generation of exoskeletons means a need to implement new hardware paradigms, and to enhance control strategies focused on assist-as-needed scheme. Finally, some aspects to improve current designs of the exoskeleton are presented.

One of the main challenges of current exoskeletons is the adaptation to the patients' walking capabilities. The proposal of a new device made from modular joints actuators offers new ways of adaptability. However, it also requires changes in control techniques. In this contribution, we introduce the use of a central pattern generator (CPG) based on adaptive Hopf oscillators in order to achieve the decentralised architecture for the modular exoskeleton. Hebbian learning algorithm is used to train the Adaptive CPG, using the data collected from a healthy subject's gait to mimic the human walking trajectories on the hip and knee. The analysis shows how the oscillator manages to learn the training signal and proves that once the oscillator has completed the learning process it no longer needs this signal. Furthermore, its modulation properties of amplitude and frequency are demonstrated, turning out to be suitable in the exoskeletons' control. Finally, results show how the algorithm is tested in two different configurations of the modular exoskeleton prototype, confirming its functioning.

Purpose To analyse the versatility and adaptability of commercially available exoskeletons for mobility assistance and their adaptation to diverse pathologies through a review of clinical trials in robotic lower limb training. Data sources A computer-aided search in bibliographic databases (PubMed and Web of Science) of clinical trials published up to September 2020 was done. Methods To be selected for detailed review, clinical trials had to meet the following criteria: (1) a protocol was designed and approved, (2) participants were people with pathologies, and (3) the trials were not a single case study. Clinical trial data were collected, extracted, and analysed, considering: objectives, trial participants, number of sessions, pathologies involved, and conclusions. Results The search resulted in 312 potentially relevant studies of seven commercial exoskeletons, of which 135 passed the preliminary screening; and 69 studies were finally selected. Of the 69 clinical trials included in the review about 50% involved Spinal Cord Injury participants, while roughly 25% focussed on stroke and two trials corresponded to patients with both disorders. The rest were composed of neurological diseases and trauma disorders. Conclusions The use of a single wearable robot for different medical conditions in various diseases is a challenge. Based on this comparative, the properties of the exoskeletons that improve the working ability with different pathologies and patient conditions have been evaluated. Suggestions were made for developing a new lower-limb exoskeleton based on various modules with a distributed control system to improve versatility in wearable technology for different gait pattern progression. Implications for rehabilitation Wearable robotic exoskeletons for gait assistance have been analysed from the perspective of adaptation to different diseases. This paper emphasizes the importance of personalized therapies and adaptive assistive technology. Suggestions were made for a new modular exoskeleton capable of addressing the issue of low versatility characterizing currently wearable assistive technology.

Preparations of comfrey (Symphytum officinale L.) roots are used topically to reduce inflammation. Comfrey anti-inflammatory and analgesic properties have been proven in clinical studies. However, the bioactive compounds associated with these therapeutic activities are yet to be identified. An LC&ndash, ESI&ndash, Orbitrap&ndash, MSn metabolite profile of a hydroalcoholic extract of comfrey root guided the identification of 20 compounds, including a new arylnaphthalene lignan bearing a rare &delta, lactone ring, named comfreyn A. Its structure was determined using extensive 2D NMR and ESI&ndash, MS experiments. Additionally, the occurrence of malaxinic acid, caffeic acid ethyl ester, along with the lignans ternifoliuslignan D, 3-carboxy-6,7-dihydroxy-1-(3&prime, 4&prime, dihydroxyphenyl) -naphthalene, globoidnan A and B, and rabdosiin was reported in S. officinale for the first time. These results helped to redefine the metabolite profile of this medicinal plant. Finally, caffeic acid ethyl ester and comfreyn A were found to significantly inhibit E-selectin expression in IL-1&beta, stimulated human umbilical vein endothelial cells (HUVEC), with EC values of 64 and 50 &micro, M, respectively.

Analysis of the genome sequence of the myxobacterium Chondromyces crocatus Cm c5 revealed the presence of numerous cryptic megasynthetase gene clusters, one of which we here assign to two previously unknown chlorinated metabolites by a comparative gene inactivation and secondary metabolomics approach. Structure elucidation of these compounds revealed a unique cyclic depsipeptide skeleton featuring β- and δ-amide bonds of aspartic acid and 3-methyl ornithine moieties, respectively. Insights into their biosynthesis were obtained by targeted gene inactivation and feeding experiments employing isotope-labeled precursors. The compounds were produced ubiquitously by the species Chondromyces crocatus and were found to inhibit the carbon storage regulator-RNA interaction.

The structures of five linear lipopeptides, thaxteramides A1, A2, B1, B2, and C isolated from the myxobacterium

Resumen: Objetivo. Determinar valores hematológicos en Ovinos Criollos de Pelo hembras durante la fase de gestación en el departamento de Córdoba. Materiales y métodos. Se analizaron 74 muestras de sangre extraídas de la vena yugular en tubos BD Vacutainer* al vacío con anticoagulante EDTA de ovejas gestantes divididas en 4 grupos (91 días de gestación) a las que se les realizaron hemogramas con un analizador automático. Resultados. Los resultados de la presente investigación para las variables hematológicas de ovejas criollas colombianas no presentaron diferencias entre los grupos de gestación, en su mayoría, concuerdan con los reportes de otras investigaciones realizadas en razas criollas tanto de ovejas como de cabras de otros países. Los resultados muestran diferencia significativa (p 91 days of gestation) to which were made blood tests with an automatic analyzer. Results. The results of the present investigation for the hematological variables of Colombian Creole sheep did not show differences between the gestation groups, in their majority, they agree with the reports of other investigations carried out in Creole breeds of sheep and goats from other countries. Te results show a significant difference (p

In our screening efforts to identify unique scaffolds from myxobacteria for the drug discovery process, we used LC-SPE-NMR-MS techniques to isolate six linear peptides, termed macyranone A-F, from Cystobacter fuscus MCy9118. The macyranones are characterized by a rare 2-methylmalonamide moiety and an α-amino ketone fragment including an α',β'-epoxyketone in macyranone A. Gene disruption experiments confirmed the biosynthetic gene cluster of the macyranones as PKS/NRPS hybrid. Detailed in silico and phylogenetic analysis unraveled that the biosynthesis involves two conspicuous amide bond formations accomplished by an amidotransferase and a unique condensation domain. The gene cluster provides further insights into the formation of the powerful epoxyketone residue involving an acyl-CoA dehydrogenase and an unconventional free-standing thioesterase. Macyranone A was found to inhibit the chymotrypsin-like activity of the yeast 20S proteasome with an IC50 of 5.9 nM and the human constitutive proteasome and immunoproteasome with IC50 values of 21 and 15 nM, respectively. The β5 subunit of the 20S proteasome was characterized as target by X-ray crystallography revealing an irreversible binding mode similar to the natural product epoxomicin. The presence of the methylmalonamide residue facilitates the stabilization of macyranone A with the active β5 subunit of the proteasome. Macyranone A exhibits a potent inhibitory effect against the parasites Trypanosoma brucei rhodesiense and Leishmania donovani with IC50 values of 1.55 and 0.22 μM, respectively.

Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed ccdB counterselection combined with oligonucleotide-mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from Photorabdus luminescens directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene plu3263 in E. coli led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site-directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi-copy plasmids.

Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-D-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.

Three new siderophores, termed hyalachelins A-C (1-3), were isolated from the terrestrial myxobacterium Hyalangium minutum. Their structures were determined by 2D NMR and HR-MS/MS experiments, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and circular dichroism experiments. Hyalachelins are unusual catecholate-type siderophores that bear a 3,7,8-trihydroxy-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Their iron chelating activities were evaluated in a CAS assay showing EC50 values of ∼30 μM.

Cystomanamides A-D were isolated as novel natural product scaffolds from Cystobacter fuscus MCy9118, and their structures were established by spectroscopic techniques including 2D NMR, LC-SPE-NMR/-MS, and HR-MS. The cystomanamides contain β-hydroxy amino acids along with 3-amino-9-methyldecanoic acid that is N-glycosylated in cystomanamide C and D. The gene cluster for cystomanamide biosynthesis was identified by gene disruption as PKS/NRPS hybrid incorporating an iso-fatty acid as starter unit and including a reductive amination step at the interface of the PKS and NRPS modules.

The present work deals with the optimization of an inhibitor of PqsD, an enzyme essential for Pseudomonas aeruginosa quorum sensing apparatus. Molecular docking studies, supported by biophysical methods (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR), were used to illuminate the binding mode of the 5-aryl-ureidothiophene-2-carboxylic acids. Enabled to make profound predictions, structure-based optimization led to increased inhibitory potency. Finally a covalent inhibitor was obtained. Binding to the active site was confirmed by LC-ESI-MS and MALDI-TOF-MS experiments. Following this rational approach, potent PqsD inhibitors were efficiently developed within a short period of time. This example shows that a combination and careful application of in silico and biophysical methods represents a powerful complement to cocrystallography.

Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.

Seven new halogenated peptides termed svetamycins A-G (1-7) have been isolated from laboratory cultures of a Streptomyces sp. Svetamycins A-D, F, and G are cyclic depsipeptides, whereas svetamycin E is a linear analogue of svetamycin C. Their structures were determined using extensive spectroscopic analysis, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and chemical derivatizations. Svetamycins are characterized by the presence of a hydroxyl acetic acid and five amino acids including a rare 4,5-dihydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid, a g-halogenated piperazic acid, and a novel d-methylated piperazic acid in svetamycins B-C, E, and G. Moreover, isotope-labeled substrate feeding experiments demonstrated ornithine as the precursor of piperazic acid and that methylation at the d position of the piperazyl scaffold is S-adenosyl-l-methionine (SAM)-dependent. Svetamycin G, the most potent antimicrobial of this suite of compounds, inhibited the growth of Mycobacterium smegmatis with an MIC80 value of 2 mg/mL.

A new linear polyketide, juniperolide A (1), was produced by the terrestrial actinomycete (Lv1-48) isolated from the rhizosphere of the plant Juniperus excelsa. The juniperolide A (1) structure contains a THP unit and a 3-amino-2,3,6-trideoxyhexose as the glycosidic moiety. Mosher's analysis was used for absolute stereochemistry determinations at C-2, C-8, C-20, and C-4', while the relative stereochemistry assignments of the remaining stereocenters were based on ROESY correlations and J-based coupling.

A new sulfated cyclic depsipeptide, termed mutremdamide A, and six new highly N-methylated peptides, termed koshikamides C-H, were isolated from different deep-water specimens of Theonella swinhoei and Theonella cupola. Their structures were determined using extensive 2D NMR, ESI, or CDESI and QTOF-MS/MS experiments and absolute configurations established by quantum mechanical calculations, advanced Marfey's method, and chiral HPLC. Mutremdamide A displays a rare 2-amino-3-(2-hydroxyphenyl)propanoic acid and a new N(delta)-carbamoyl-beta-sulfated asparagine. Koshikamides C-E are linear undecapeptides, and koshikamides F-H are 17-residue depsipeptides containing a 10-residue macrolactone. Koshikamides F and G differ from B and H in part by the presence of the conjugated unit 2-(3-amino-5-oxopyrrolidin-2-ylidene)propanoic acid. Cyclic koshikamides F and H inhibited HIV-1 entry at low micromolar concentrations while their linear counterparts were inactive. The Theonella collections studied here are distinguished by co-occurrence of mutremdamide A, koshikamides, and theonellamides, the combination of which appears to define a new Theonella chemotype that can be found in deeper waters.

Three new anabaenopeptin-like peptides, named paltolides A-C, were isolated from a deep-water specimen of the marine sponge Theonella swinhoei from Palau. Paltolides belong to a rare subgroup of sponge-derived anabaenopeptins that have in common a C-terminal tryptophan residue linked to the epsilon-amine of a lysine bearing a d configuration. The structures of paltolides A-C were determined by NMR and tandem MS techniques. Paltolide A is the first anabaenopeptin structure where a non-N-methylated amino acid precedes the C-terminal residue.

A new highly unsaturated macrolide lactam, termed mirabilin ( 1), was isolated from the aqueous extract of the marine sponge Siliquariaspongia mirabilis. Mirabilin is characterized by the presence of a 35-membered macrolide lactam ring bearing a pentadiene conjugated system and a tetrasubstituted tetrahydropyran ring. A linear polyketide moiety is attached to the macrocyclic ring through an amide linkage. The structure of mirabilin was determined using extensive 2D NMR and ESIMS and tandem MS techniques. Mirabilin inhibits the growth of the tumor cell line HCT-116 with an IC 50 value of 0.27 +/- 0.09 microM and is noncytotoxic to several other cell lines.

Seven new chromone-containing polyketides, termed cystochromones A-G, were isolated from the myxobacterial strain Cystobacter sp. MCy9104. Their structures were elucidated using comprehensive NMR spectroscopy and HR-MS/MS. Cystochromones bear a pentadecyl moiety unusually attached at C-5 of the chromone ring. Moreover, isotope-labeled substrate feeding experiments and NMR analysis suggested a hybrid iso-fatty acid and polyketide synthase biosynthetic pathway for these secondary metabolites.

Five new 14,15-secopregnane derivatives, named argelosides K-O (1-5), have been isolated from Solenostemma argel leaves. Their structures were established by a detailed spectroscopic analysis. In particular, argeloside N (1) showed in the sugar portion an unusual 3-O-methyl-2,6-dideoxyhexopyranose unit characterized by the occurrence of a Delta3 double bond, and argeloside O (5) displayed an unusual moiety linked to position 3 probably derived by the oxidation of a 3-O-methyl-2,6-dideoxyhexopyranose unit. The propensity of compounds 2, 3, and 5 and argeloside F (6) to inhibit TNF-alpha release by LPS-stimulated RAW 264.7 mouse cells was evaluated.

Seven new 15-keto pregnane glycosides, namely Stemmosides E--K, were isolated from Solenostemma argel. Stemmosides E--J are characterized by the occurrence of an uncommon 14 beta proton configuration while stemmosides E and F possess in addition a rare enolic function in C-16. On the other hand, stemmosides G-J display an unusual C-17 alpha side chain. Their structures were established by ESI-MS and NMR experiments. Moreover, the effect of these compounds on the VEGF-induced in Kaposi's sarcoma cell proliferation was tested. Results indicated that all the compounds reduced the cell proliferation in a dose dependent manner.

Two new dammarane saponins identified as jujubogenin 3-O-alpha-l-arabinofuranosyl(1-->2)-[beta-d-glucopyranosyl(1-->6) beta-d-glucopyranosyl(1-->3)]-alpha-l-arabinopyranoside (2) and jujubogenin 3-O-alpha-l-arabinofuranosyl(1-->2)-[6-O-[3-hydroxy-3-methylglutaryl]-beta-d-glucopyranosyl(1-->3)]-alpha-l-arabinopyranoside (3) and a new lupane saponin, 3beta-hydroxylup-20(29)-en-27,28-dioic acid 28-O-beta-d-glucopyranosyl(1-->2)-[beta-d-xylopyranosyl(1-->3)]-beta-d-xylopyranosyl(1-->2)-beta-d-glucopyranoside ester (5), along with the known jujubogenin 3-O-alpha-l-arabinofuranosyl(1-->2)-[beta-d-glucopyranosyl(1-->3)]-alpha-l-arabinopyranoside (1) and 3beta-hydroxylup-20(29)-ene-27,28-dioic acid (4), were isolated from the methanol extract of the stems of Anomospermum grandifolium. The structures of the new compounds were established by spectral analysis. Antimicrobial activity screening of compounds 1-3 revealed antifungal properties against C. albicans ATCC 3153 for compounds 2 and 3. The antibacterial and antifungal activities of the petroleum ether, chloroform, and methanol extracts of A. grandifolium stems were also evaluated.

Biosynthetic pathway intermediates related to genotoxin colibactin formation: a linear compound 3 bearing a rare 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue., Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-d-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.

We assessed in vivo the anti-inflammatory activity of two Cat's claw bark extracts, by comparing a spray-dried hydroalcoholic extract against an aqueous freeze-dried extract, to determine which extract was more effective. We used the carrageenan-induced paw edema model in mice. In addition, to assess the molecular mechanism of action, we determined the inhibition of NF-kappa B through the Electrophoretic Mobility Shift Assay (EMSA) and the effects on cycloxygenase-1 and -2. Results showed that the anti-inflammatory activity was significantly higher using the hydroalcoholic compared with the aqueous extract (P

Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed ccdB counterselection combined with oligonucleotide-mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from Photorabdus luminescens directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene plu3263 in E. coli led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site-directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi-copy plasmids.

Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant Staphylococcus aureus (MRSA) strains (MIC 2-16 μg mL(-1)) and rifampicin-resistant Escherichia coli TolC strains (MIC 12.5-50 μg mL(-1)). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (Myx) suggest that the ureidothiophene-2-carboxylic acids compete with Myx for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the Myx western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance.