Microwave-Assisted Extraction of Secondary Metabolites Using Ethyl Lactate Green Solvent from Ambrosia arborescens: LC/ESI-MS/MS and Antioxidant Activity.

Alternative solvents are being tested as green solvents to replace the traditional organic solvents used in both academy and industry. Some of these are already available, such as ethyl lactate, cyrene, limonene, glycerol, and others. This alternative explores eco-friendly processes for extracting secondary metabolites from nature, thus increasing the number of unconventional extraction methods with lower environmental impact over conventional methods. In this context, the Peruvian Ambrosia arborescens was our model while exploring a microwave-assisted extraction (MAE) approach over maceration. The objective of this study was to perform a phytochemical study including UHPLC-ESI-MS/MS and the antioxidant activity of Ambrosia arborescens, using sustainable strategies by mixing both microwaves and ethyl lactate as a green solvent. The results showed that ethyl lactate/MAE (15.07%) achieved a higher extraction yield than methanol/maceration (12.6%). In the case of the isolation of psilostachyin, it was similar to ethyl lactate (0.44%) when compared to methanol (0.40%). Regarding UHPLC-ESI-MS/MS studies, the results were similar. Twenty-eight compounds were identified in the ethyl lactate/MAE and methanol/maceration extracts, except for the tentative identification of two additional amino acids (peaks 4 and 6) in the MeOH extract. In relation to the antioxidant assay, the activity of the ethyl lactate extract was a little higher than the methanol extract in terms of ORAC (715.38 ± 3.2) and DPPH (263.04 ± 2.8). This study on A. arborescens demonstrated that the unconventional techniques, such as MAE related to ethyl lactate, could replace maceration/MeOH for the extraction and isolation of metabolites from diverse sources. This finding showed the potential of unconventional methods with green solvents to provide eco-friendly methods based on green chemistry.

Plasmodium knowlesi Infection in Traveler Returning to Canada from the Philippines, 2023.

A 55-year-old man sought treatment for an uncomplicated febrile illness after returning to Canada from the Philippines. A suspected diagnosis of Plasmodium knowlesi infection was confirmed by PCR, and treatment with atovaquone/proguanil brought successful recovery. We review the evolving epidemiology of P. knowlesi malaria in the Philippines, specifically within Palawan Island.

Fatty Acid Substitutions Modulate the Cytotoxicity of Puwainaphycins/Minutissamides Isolated from the Baltic Sea Cyanobacterium Nodularia harveyana UHCC-0300.

Puwainaphycins (PUW) and minutissamides (MIN) are structurally homologous cyclic lipopeptides that exhibit high structural variability and possess antifungal and cytotoxic activities. While only a minor variation can be found in the amino acid composition of the peptide cycle, the fatty acid (FA) moiety varies largely. The effect of FA functionalization on the bioactivity of PUW/MIN chemical variants is poorly understood. A rapid and selective liquid chromatography-mass spectrometry-based method led us to identify 13 PUW/MIN (1-13) chemical variants from the benthic cyanobacterium Nodularia harveyana strain UHCC-0300 from the Baltic Sea. Five new variants identified were designated as PUW H (1), PUW I (2), PUW J (4), PUW K (10), and PUW L (13) and varied slightly in the peptidic core composition, but a larger variation was observed in the oxo-, chloro-, and hydroxy-substitutions on the FA moiety. To address the effect of FA substitution on the cytotoxic effect, the major variants (3 and 5-11) together with four other PUW/MIN variants (14-17) previously isolated were included in the study. The data obtained showed that hydroxylation of the FA moiety abolishes the cytotoxicity or significantly reduces it when compared with the oxo-substituted C18-FA (compounds 5-8). The oxo-substitution had only a minor effect on the cytotoxicity of the compound when compared to variants bearing no substitution. The activity of PUW/MIN variants with chlorinated FA moieties varied depending on the position of the chlorine atom on the FA chain. This study also shows that variation in the amino acids distant from the FA moiety (position 4-8 of the peptide cycle) does not play an important role in determining the cytotoxicity of the compound. These findings confirmed that the lipophilicity of FA is essential to maintain the cytotoxicity of PUW/MIN lipopeptides. Further, a 63 kb puwainaphycin biosynthetic gene cluster from a draft genome of the N. harveyana strain UHCC-0300 was identified. This pathway encoded two specific lipoinitiation mechanisms as well as enzymes needed for the modification of the FA moiety. Examination on biosynthetic gene clusters and the structural variability of the produced PUW/MIN suggested different mechanisms of fatty-acyl-AMP ligase cooperation with accessory enzymes leading to a new set of PUW/MIN variants bearing differently substituted FA.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Establishing Healthcare Worker Performance and Safety in Providing Critical Care for Patients in a Simulated Ebola Treatment Unit: Non-Randomized Pilot Study.

Improving the provision of supportive care for patients with Ebola is an important quality improvement initiative. We designed a simulated Ebola Treatment Unit (ETU) to assess performance and safety of healthcare workers (HCWs) performing tasks wearing personal protective equipment (PPE) in hot (35 °C, 60% relative humidity) or thermo-neutral (20 °C, 20% relative humidity) conditions. In this pilot phase to determine the feasibility of study procedures, HCWs in PPE were non-randomly allocated to hot or thermo-neutral conditions to perform peripheral intravenous (PIV) and midline catheter (MLC) insertion and endotracheal intubation (ETI) on mannequins. Eighteen HCWs (13 physicians, 4 nurses, 1 nurse practitioner; 2 with prior ETU experience; 10 in hot conditions) spent 69 (10) (mean (SD)) minutes in the simulated ETU. Mean (SD) task completion times were 16 (6) min for PIV insertion; 33 (5) min for MLC insertion; and 16 (8) min for ETI. Satisfactory task completion was numerically higher for physicians vs. nurses. Participants' blood pressure was similar, but heart rate was higher (p = 0.0005) post-simulation vs. baseline. Participants had a median (range) of 2.0 (0.0-10.0) minor PPE breaches, 2.0 (0.0-6.0) near-miss incidents, and 2.0 (0.0-6.0) health symptoms and concerns. There were eight health-assessment triggers in five participants, of whom four were in hot conditions. We terminated the simulation of two participants in hot conditions due to thermal discomfort. In summary, study tasks were suitable for physician participants, but they require redesign to match nurses' expertise for the subsequent randomized phase of the study. One-quarter of participants had a health-assessment trigger. This research model may be useful in future training and research regarding clinical care for patients with highly infectious pathogens in austere settings.

Structure elucidation of the novel carotenoid gemmatoxanthin from the photosynthetic complex of Gemmatimonas phototrophica AP64.

Gemmatimonas phototrophica AP64 is the first phototrophic representative of the bacterial phylum Gemmatimonadetes. The cells contain photosynthetic complexes with bacteriochlorophyll a as the main light-harvesting pigment and an unknown carotenoid with a single broad absorption band at 490 nm in methanol. The carotenoid was extracted from isolated photosynthetic complexes, and purified by liquid chromatography. A combination of nuclear magnetic resonance (1H NMR, COSY, 1H-13C HSQC, 1H-13C HMBC, J-resolved, and ROESY), high-resolution mass spectroscopy, Fourier-transformed infra-red, and Raman spectroscopy was used to determine its chemical structure. The novel linear carotenoid, that we have named gemmatoxanthin, contains 11 conjugated double bonds and is further substituted by methoxy, carboxyl and aldehyde groups. Its IUPAC-IUBMB semi-systematic name is 1'-Methoxy-19'-oxo-3',4'-didehydro-7,8,1',2'-tetrahydro- Ψ, Ψ carotene-16-oic acid. To our best knowledge, the presence of the carboxyl, methoxy and aldehyde groups on a linear C40 carotenoid backbone is reported here for the first time.

Cyanochelins, an Overlooked Class of Widely Distributed Cyanobacterial Siderophores, Discovered by Silent Gene Cluster Awakening.

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring ß-hydroxyaspartate (ß-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of ß-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe3+ to Fe2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread ß-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, ß-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Semi-synthetic puwainaphycin/minutissamide cyclic lipopeptides with improved antifungal activity and limited cytotoxicity.

Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 µM for MIN A and 3.2 µM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 µM) and the plant pathogen Alternaria alternata (0.6 µM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 µM) and A. alternata (MIC = 0.1-0.5 µM), but with partial retention of the cytotoxic effect (∼10-20 µM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 µM), and greatly reduced or abolished cytotoxic activity (>20 µM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.

Cyanobacterial lipopeptides puwainaphycins and minutissamides induce disruptive and pro-inflammatory processes in Caco-2 human intestinal barrier model.

Puwainaphycins (PUW) and minutissamides (MIN) are cyanobacterial lipopeptides found in various cyanobacterial species. The first possible target of human exposure to them is intestinal epithelium but effect of PUW/MIN on enterocytes is not known at all. Using differentiated Caco-2 cells, PUW F was found to be cytotoxic from 5 µM concentration based on lactate dehydrogenase release assay and total protein concentration. However, it is also able to induce production of interleukin 8 in non-cytotoxic concentrations 1 and 2.5 µM detected by ELISA. Effects of MIN A and C were similar but less pronounced compared to PUW F. On the other hand, MIN D was the least toxic compound with no significant pro-inflammatory effects. Surprisingly, pro-inflammatory activation of the cells by PUW F and MIN C resulted in an increase in tight junction (TJ) protein claudin 4 expression determined by western blot analysis and confirmed by confocal microscopy. Furthermore, decrease in expression of zonula occludens 3, another TJ protein, was observed after the exposure to PUW F. Taken together, these cytotoxic lipopeptides, especially PUW F, are to be studied more deeply due to their capability to activate and/or deregulate human enterocytes in low concentrations.

Atypical clinical presentation of Ebola virus disease in pregnancy: Implications for clinical and public health management.

BACKGROUND: Between December 2013 and June 2016, West Africa experienced the largest Ebola virus disease (EVD) outbreak in history. Understanding EVD in pregnancy is important for EVD clinical screening and infection prevention and control. METHODS: We conducted a review of medical records and EVD investigation reports from three districts in Sierra Leone. We report the clinical presentations and maternal and fetal outcomes of six pregnant women with atypical EVD, and subsequent transmission events from perinatal care. RESULTS: The six women (ages 18-38) were all in the third trimester. Each presented with signs and symptoms initially attributed to pregnancy. None met EVD case definition; only one was known at presentation to be a contact of an EVD case. Five women died, and all six fetuses/neonates died. These cases resulted in at least 35 additional EVD cases. CONCLUSIONS: These cases add to the sparse literature focusing on pregnant women with EVD, highlighting challenges and implications for outbreak control. Infected newborns may also present atypically and may shed virus while apparently asymptomatic. Pregnant women identified a priori as contacts of EVD cases require special attention and planning for obstetrical care.

Antimicrobial activity and bioactive profiling of heterocytous cyanobacterial strains using MS/MS-based molecular networking.

The rapid emergence of resistance in pathogenic bacteria together with a steep decline in economic incentives has rendered a new wave in the drug development by the pharmaceutical industry and researchers. Since cyanobacteria are recognized as wide producers of pharmaceutically important compounds, we investigated thirty-four cyanobacterial extracts prepared by solvents of different polarities for their antimicrobial potential. Almost all tested cyanobacterial strains exhibited some degree of antimicrobial bioactivity, with more general effect on fungal strains compared with bacteria. Surprisingly ~50% of cyanobacterial extracts exhibited specific activity against one or few bacterial indicator strains with Gram-positive bacteria being more affected. Extracts of two most promising strains were subjected to activity-guided fractionation and determination of the minimum inhibitory concentration (MIC) against selected bacterial and fungal isolates. Multiple fractions were responsible for their antimicrobial effect with MIC reaching low-micromolar concentrations and in some of them high level of specificity was recorded. Twenty-six bioactive fractions analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) online workflow using dereplication resulted in identification of only forty-nine peptide spectrum matches (PSMs) with eleven unique metabolites spectrum matches (MSMs). Interestingly, only three fractions from Nostoc calcicola Lukesová 3/97 and four fractions from Desmonostoc sp. Cc2 showed the presence of unique MSMs suggesting the presence of unknown antimicrobial metabolites among majority of bioactive fractions from both the strains. Our results highlight potential for isolation and discovery of potential antimicrobial bioactive lead molecules from cyanobacterial extracts.

Anti-Ebola therapy for patients with Ebola virus disease: a systematic review.

BACKGROUND: Management of Ebola virus disease (EVD) has historically focused on infection prevention, case detection and supportive care. Several specific anti-Ebola therapies have been investigated, including during the 2014-2016 West African outbreak. Our objective was to conduct a systematic review of the effect of anti-Ebola virus therapies on clinical outcomes to guide their potential use and future evaluation. METHODS: We searched PubMed, EMBASE, Global Health, Cochrane Library, African Index Medicus, WHOLIS (inception-9 April 2018), and trial registries for observational studies or clinical trials, in any language, that enrolled patients with confirmed EVD who received therapy targeting Ebola virus and reported on mortality, symptom duration, or adverse effects. RESULTS: From 11,257 citations and registered trials, we reviewed 55 full-text citations, of which 35 met eligibility criteria (1 randomized clinical trial (RCT), 8 non-randomized comparative studies, 9 case series and 17 case reports) and collectively examined 21 anti-Ebola virus agents. The 31 studies performed during the West African outbreak reported on 4.8% (1377/28616) of all patients with Ebola. The only RCT enrolled 72 patients (0.25% of all patients with Ebola) and compared the monoclonal antibody ZMapp vs. standard care (mortality, 22% vs. 37%; 95% confidence interval for risk difference, - 36 to 7%). Studies of convalescent plasma, interferon-ß-1a, favipiravir, brincidofovir, artesunate-amodiaquine and TKM-130803 were associated with at least moderate risk of bias. CONCLUSIONS: Research evaluating anti-Ebola virus agents has reached very few patients with EVD, and inferences are limited by non-randomized study designs. ZMapp has the most promising treatment signal.

Priorities, Barriers, and Facilitators towards International Guidelines for the Delivery of Supportive Clinical Care during an Ebola Outbreak: A Cross-Sectional Survey.

During the Ebola outbreak, mortality reduction was attributed to multiple improvements in supportive care delivered in Ebola treatment units (ETUs). We aimed to identify high-priority supportive care measures, as well as perceived barriers and facilitators to their implementation, for patients with Ebola Virus Disease (EVD). We conducted a cross-sectional survey of key stakeholders involved in the response to the 2014⁻2016 West African EVD outbreak. Out of 57 email invitations, 44 responses were received, and 29 respondents completed the survey. The respondents listed insufficient numbers of health workers (23/29, 79%), improper tools for the documentation of clinical data (n = 22/28, 79%), insufficient material resources (n = 22/29, 76%), and unadapted personal protective equipment (n = 20/28, 71%) as the main barriers to the provision of supportive care in ETUs. Facilitators to the provision of supportive care included team camaraderie (n in agreement = 25/28, 89%), ability to speak the local language (22/28, 79%), and having treatment protocols in place (22/28, 79%). This survey highlights a consensus across various stakeholders involved in the response to the 2014⁻2016 EVD outbreak on a limited number of high-priority supportive care interventions for clinical practice guidelines. Identified barriers and facilitators further inform the application of guidelines.

Spectrum of illness in migrants to Canada: sentinel surveillance through CanTravNet.

BACKGROUND: Due to ongoing political instability and conflict in many parts of the world, migrants are increasingly seeking asylum and refuge in Canada. METHODS: We examined demographic and travel correlates of illnesses among migrants to Canada to establish a detailed epidemiologic framework of this population for Canadian practitioners. Data on ill-returned Canadian travellers presenting to a CanTravNet site between 1 January 2015 and 31 December 2015 were analyzed. RESULTS: During the study period, 2415 ill travellers and migrants presented to a CanTravNet site, and of those, 519 (21.5%) travelled for the purpose of migration. Sub-Saharan Africa (n = 160, 30.8%), southeast Asia (n = 84, 16.2%) and south central Asia (n = 75, 14.5%) were the most common source regions for migrants, while the top specific source countries, of 98 represented, were the Philippines (n = 45, 8.7%), China (n = 36, 6.9%) and Vietnam (n = 31, 6.0%). Compared with non-migrant travellers, migrants were more likely to have a pre-existing immunocompromising medical condition, such as HIV or diabetes mellitus (P < 0.0001), and to require inpatient management of their illness (P < 0.0001). Diagnoses such as tuberculosis (n = 263, 50.7%), hepatitis B and C (n = 78, 15%) and HIV (n = 11, 2.1%) were over-represented in the migrant population compared with non-migrant travellers (P < 0.0001). Most cases of tuberculosis in the migrant population (n = 263) were latent (82% [n = 216]); only 18% (n = 47) were active. CONCLUSIONS: Compared with non-migrant travellers, migrants were more likely to present with a communicable infectious disease, such as tuberculosis, potentially complicated by an underlying immunosuppressing condition such as HIV. These differences highlight the divergent healthcare needs in the migrant population, and underscore the importance of surveillance programmes to understand their burden of illness. Intake programming should be adequately resourced to accommodate the medical needs of this vulnerable population of new Canadians.

Herpes zoster complicated by phrenic nerve palsy and respiratory compromise.

BACKGROUND: Herpes zoster can be associated with severe neurological complications. CASE PRESENTATION: In this article, we describe the case of a 54-year-old man with herpes zoster affecting his right upper chest and neck region complicated by phrenic nerve palsy and respiratory compromise. The diagnosis of herpes zoster was made based on the classic appearance of the rash and associated neuropathic-type pain. The diagnosis of phrenic nerve palsy was made by chest x-ray and ultrasound. CONCLUSION: Clinicians should be aware of the possibility of phrenic nerve palsy occurring in patients who have herpes zoster affecting the region of C3,4,5 dermatomes. Although symptoms of unilateral diaphragmatic paresis are usually mild, in patients with obesity or comorbid lung disease, new onset phrenic nerve palsy can lead to significant respiratory compromise.

A community-based innovative model of care for the management of severe bacterial infections in persons who use injection drugs.

The opioid crisis and complications related to injection drug use are a public health emergency. The combination of addiction and injection drug use is a devastating double-edged sword: it predisposes patients to severe life-threatening infections like endocarditis, and epidural abscess, as well as to disorganized behaviour and impaired decision-making that interferes with the completion of prolonged courses of required antibiotic therapy. Poverty and stigma add further fuel to the fire. The Community Transitional Care Team (CTCT) is a revolutionary community-based short-term residence where people who inject drugs can stay to complete their course of antibiotics. We present the case of a young woman struggling with addiction, tremendous social barriers to health, and life threating Staphylococcus aureus infection that highlights the benefits of strong, community-based, and individualized models of patient care.

La crise des opioïdes et les complications liées à la consommation de drogues injectables représentent une urgence de santé publique. L'association de la dépendance et de la consommation de drogues injectables est une épée à double tranchant dévastatrice. En effet, elle prédispose les patients à de graves infections au potentiel mortel comme l'endocardite et l'abcès épidural, de même qu'à une désorganisation du comportement et à une altération des décisions qui compromettent les antibiothérapies prolongées. La pauvreté et l'opprobre jettent de l'huile sur le feu. L'équipe communautaire de soins transitoires est une résidence communautaire de courte durée révolutionnaire où les personnes qui s'injectent des drogues peuvent habiter pour terminer leur antibiothérapie. Les auteurs présentent le cas d'une jeune femme aux prises avec la dépendance, d'énormes obstacles sociaux en matière de santé et une infection à Staphylococcus aureus au potentiel mortel qui fait ressortir les avantages de modèles communautaires et personnalisés robustes pour les soins aux patients.

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides.

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

Application of HPCCC Combined with Polymeric Resins and HPLC for the Separation of Cyclic Lipopeptides Muscotoxins A⁻C and Their Antimicrobial Activity.

Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1⁻3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2⁻3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.