New myxobacteria of the Myxococcaceae clade produce angiolams with antiparasitic activities.

In the past century, microbial natural products have proven themselves to be substantial and fruitful sources of anti-infectives. In addition to the well-studied Actinobacteria, understudied bacterial taxa like the Gram-negative myxobacteria have increasingly gained attention in the ongoing search for novel and biologically active natural products. In the course of a regional sampling campaign to source novel myxobacteria, we recently uncovered new myxobacterial strains MCy12716 and MCy12733 belonging to the Myxococcaceae clade. Early bioactivity screens of the bacterial extracts revealed the presence of bioactive natural products that were identified as angiolam A and several novel derivatives. Sequencing of the corresponding producer strains allowed the identification of the angiolam biosynthetic gene cluster, which was verified by targeted gene inactivation. Based on bioinformatic analysis of the biosynthetic gene cluster, a concise biosynthesis model was devised to explain angiolam biosynthesis. Importantly, novel angiolam derivatives uncovered in this study named angiolams B, C, and D were found to display promising antiparasitic activities against the malaria pathogen Plasmodium falciparum in the 0.3-0.8 µM range.IMPORTANCEThe COVID-19 pandemic and continuously emerging antimicrobial resistance (AMR) have recently raised awareness about limited treatment options against infectious diseases. However, the shortage of treatment options against protozoal parasitic infections, like malaria, is much more severe, especially for the treatment of so-called neglected tropical diseases. The detection of anti-parasitic bioactivities of angiolams produced by MCy12716 and MCy12733 displays the hidden potential of scarcely studied natural products to have promising biological activities in understudied indications. Furthermore, the improved biological activities of novel angiolam derivatives against Plasmodium falciparum and the evaluation of its biosynthesis display the opportunities of the angiolam scaffold on route to treat protozoal parasitic infections as well as possible ways to increase the production of derivatives with improved bioactivities.

Temperature-Dependent Selection of Reaction Pathways, Reactive Species, and Products during Postsynthetic Selenization of Copper Sulfide Nanoparticles.

Rational design of elaborate, multicomponent nanomaterials is important for the development of many technologies such as optoelectronic devices, photocatalysts, and ion batteries. Combination of metal chalcogenides with different anions, such as in CdS/CdSe structures, is particularly effective for creating heterojunctions with valence band offsets. Seeded growth, often coupled with cation exchange, is commonly used to create various core/shell, dot-in-rod, or multipod geometries. To augment this library of multichalcogenide structures with new geometries, we have developed a method for postsynthetic transformation of copper sulfide nanorods into several different classes of nanoheterostructures containing both copper sulfide and copper selenide. Two distinct temperature-dependent pathways allow us to select from several outcomes-rectangular, faceted Cu2-xS/Cu2-xSe core/shell structures, nanorhombuses with a Cu2-xS core, and triangular deposits of Cu2-xSe or Cu2-x(S,Se) solid solutions. These different outcomes arise due to the evolution of the molecular components in solution. At lower temperatures, slow Cu2-xS dissolution leads to concerted morphology change and Cu2-xSe deposition, while Se-anion exchange dominates at higher temperatures. We present detailed characterization of these Cu2-xS-Cu2-xSe nanoheterostructures by transmission electron microscopy (TEM), powder X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning TEM-energy-dispersive spectroscopy. Furthermore, we correlate the selenium species present in solution with the roles they play in the temperature dependence of nanoheterostructure formation by comparing the outcomes of the established reaction conditions to use of didecyl diselenide as a transformation precursor.

Causal influence of brainstem response to transcutaneous vagus nerve stimulation on cardiovagal outflow.

BACKGROUND: The autonomic response to transcutaneous auricular vagus nerve stimulation (taVNS) has been linked to the engagement of brainstem circuitry modulating autonomic outflow. However, the physiological mechanisms supporting such efferent vagal responses are not well understood, particularly in humans. HYPOTHESIS: We present a paradigm for estimating directional brain-heart interactions in response to taVNS. We propose that our approach is able to identify causal links between the activity of brainstem nuclei involved in autonomic control and cardiovagal outflow. METHODS: We adopt an approach based on a recent reformulation of Granger causality that includes permutation-based, nonparametric statistics. The method is applied to ultrahigh field (7T) functional magnetic resonance imaging (fMRI) data collected on healthy subjects during taVNS. RESULTS: Our framework identified taVNS-evoked functional brainstem responses with superior sensitivity compared to prior conventional approaches, confirming causal links between taVNS stimulation and fMRI response in the nucleus tractus solitarii (NTS). Furthermore, our causal approach elucidated potential mechanisms by which information is relayed between brainstem nuclei and cardiovagal, i.e., high-frequency heart rate variability, in response to taVNS. Our findings revealed that key brainstem nuclei, known from animal models to be involved in cardiovascular control, exert a causal influence on taVNS-induced cardiovagal outflow in humans. CONCLUSION: Our causal approach allowed us to noninvasively evaluate directional interactions between fMRI BOLD signals from brainstem nuclei and cardiovagal outflow.

Discovery and Characterization of a Myxobacterial Lanthipeptide with Unique Biosynthetic Features and Anti-inflammatory Activity.

The genomes of myxobacteria harbor a variety of biosynthetic gene clusters encoding numerous secondary metabolites, including ribosomally synthesized and post-translationally modified peptides (RiPPs) with diverse chemical structures and biological activities. However, the biosynthetic potential of RiPPs from myxobacteria remains barely explored. Herein, we report a novel myxobacteria lanthipeptide myxococin identified from Myxococcus fulvus. Myxococins represent the first example of lanthipeptides, of which the characteristic multiple thioether rings are installed by employing a Class II lanthipeptide synthetase MfuM and a Class I lanthipeptide cyclase MfuC in a cascaded way. Unprecedentedly, we biochemically characterized the first M61 family aminopeptidase MfuP involved in RiPP biosynthesis, demonstrating that MfuP showed the activity of an endopeptidase activity. MfuP is leader-independent but strictly selective for the multibridge structure of myxococin A and responsible for unwrapping two rings via amide bond hydrolysis, yielding myxococin B. Furthermore, the X-ray crystal structure of MfuP and structural analysis, including active-site mutations, are reported. Finally, myxococins are evaluated to exhibit anti-inflammatory activity in lipopolysaccharide-induced macrophages without detectable cytotoxicity.

Structure Elucidation and Biosynthesis of Nannosterols A and B, Myxobacterial Sterols from Nannocystis sp. MNa10993.

Myxobacteria represent an underinvestigated source of chemically diverse and biologically active secondary metabolites. Here, we report the discovery, isolation, structure elucidation, and biological evaluation of two new bacterial sterols, termed nannosterols A and B (1, 2), from the terrestrial myxobacterium Nannocystis sp. (MNa10993). Nannosterols feature a cholestanol core with numerous modifications including a secondary alcohol at position C-15, a terminal vicinal diol side chain at C-24-C-25 (1, 2), and a hydroxy group at the angular methyl group at C-18 (2), which is unprecedented for bacterial sterols. Another rare chemical feature of bacterial triterpenoids is a ketone group at position C-7, which is also displayed by 1 and 2. The combined exploration based on myxobacterial high-resolution secondary metabolome data and genomic in silico investigations exposed the nannosterols as frequently produced sterols within the myxobacterial suborder of Nannocystineae. The discovery of the nannosterols provides insights into the biosynthesis of these new myxobacterial sterols, with implications in understanding the evolution of sterol production by prokaryotes.

Optimization of respiratory-gated auricular vagus afferent nerve stimulation for the modulation of blood pressure in hypertension.

Background: The objective of this pilot study was to identify frequency-dependent effects of respiratory-gated auricular vagus afferent nerve stimulation (RAVANS) on the regulation of blood pressure and heart rate variability in hypertensive subjects and examine potential differential effects by sex/gender or race. Methods: Twenty hypertensive subjects (54.55 ± 6.23 years of age; 12 females and 8 males) were included in a within-person experimental design and underwent five stimulation sessions where they received RAVANS at different frequencies (i.e., 2 Hz, 10 Hz, 25 Hz, 100 Hz, or sham stimulation) in a randomized order. EKG and continuous blood pressure signals were collected during a 10-min baseline, 30-min stimulation, and 10-min post-stimulation periods. Generalized estimating equations (GEE) adjusted for baseline measures were used to evaluate frequency-dependent effects of RAVANS on heart rate, high frequency power, and blood pressure measures, including analyses stratified by sex and race. Results: Administration of RAVANS at 100 Hz had significant overall effects on the reduction of heart rate (ß = -2.03, p = 0.002). It was also associated with a significant reduction of diastolic (ß = -1.90, p = 0.01) and mean arterial blood pressure (ß = -2.23, p = 0.002) in Black hypertensive participants and heart rate in female subjects (ß = -2.83, p = 0.01) during the post-stimulation period when compared to sham. Conclusion: Respiratory-gated auricular vagus afferent nerve stimulation exhibits frequency-dependent rapid effects on the modulation of heart rate and blood pressure in hypertensive patients that may further differ by race and sex. Our findings highlight the need for the development of optimized stimulation protocols that achieve the greatest effects on the modulation of physiological and clinical outcomes in this population.

The inherent antibiotic activity of myxobacteria-derived autofluorescent outer membrane vesicles is switched on and off by light stimulation.

Outer membrane vesicles are small, lipid-based vesicles shed from the outer membrane of Gram-negative bacteria. They are becoming increasingly recognised as important factors for resistance gene transfer, bacterial virulence factors and host cell modulation. The presence of pathogenic factors and antimicrobial compounds in bacterial vesicles has been proven in recent years, but it remains unclear, if and how environmental factors, such as light specifically regulate the vesicle composition. We report the first example of autofluorescent vesicles derived from non-pathogenic soil-living myxobacteria. These vesicles additionally showed inherent antibiotic activity, a property that is specifically regulated by light stimulation of the producing bacteria. Our data provide a central basis for better understanding the environmental impact on bacteria-derived vesicles, and design of future therapeutic options.

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus Inhibitors.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

Discovery, Biosynthesis and Biological Activity of a Succinylated Myxochelin from the Myxobacterial Strain MSr12020.

Myxobacteria feature unique biological characteristics, including their capability to glide on the surface, undergo different multicellular developmental stages and produce structurally unique natural products such as the catecholate-type siderophores myxochelins A and B. Herein, we report the isolation, structure elucidation and a proposed biosynthesis of the new congener myxochelin B-succinate from the terrestrial myxobacterial strain MSr12020, featuring a succinyl decoration at its primary amine group. Myxochelin-B-succinate exhibited antibacterial growth inhibition and moderate cytotoxic activity against selected human cancer cell lines. This unique chemical modification of myxochelin B might provide interesting insights for future microbiological studies to understand the biological function and biosynthesis of secondary metabolite succinylation.

Efficacy and Safety of Vagus Nerve Stimulation on Upper Limb Motor Recovery After Stroke. A Systematic Review and Meta-Analysis.

Background: Upper limb motor impairment is one of the main complications of stroke, affecting quality of life both for the patient and their family. The aim of this systematic review was to summarize the scientific evidence on the safety and efficacy of Vagus Nerve Stimulation (VNS) on upper limb motor recovery after stroke. Methods: A systematic review and meta-analysis of studies that have evaluated the efficacy or safety of VNS in stroke patients was performed. The primary outcome was upper limb motor recovery. A search of articles published on MEDLINE, CENTRAL, EBSCO and LILACS up to December 2021 was performed, and a meta-analysis was developed to calculate the overall effects. Results: Eight studies evaluating VNS effects on motor function in stroke patients were included, of which 4 used implanted and 4 transcutaneous VNS. It was demonstrated that VNS, together with physical rehabilitation, increased upper limb motor function on average 7.06 points (95%CI 4.96; 9.16) as assessed by the Fugl-Meyer scale. Likewise, this improvement was significantly greater when compared to a control intervention (mean difference 2.48, 95%CI 0.98; 3.98). No deaths or serious adverse events related to the intervention were reported. The most frequent adverse events were dysphonia, dysphagia, nausea, skin redness, dysgeusia and pain related to device implantation. Conclusion: VNS, together with physical rehabilitation, improves upper limb motor function in stroke patients. Additionally, VNS is a safe intervention.

Corallococcus soli sp. Nov., a Soil Myxobacterium Isolated from Subtropical Climate, Chalus County, Iran, and Its Potential to Produce Secondary Metabolites.

A novel myxobacterial strain ZKHCc1 1396T was isolated in 2017 from a soil sample collected along Chalus Road connecting Tehran and Mazandaran, Iran. It was a Gram-negative, rod-shaped bacterial strain that displayed the general features of Corallococcus, including gliding and fruiting body formation on agar and microbial lytic activity. Strain ZKHCc1 1396T was characterized as an aerobic, mesophilic, and chemoheterotrophic bacterium resistant to many antibiotics. The major cellular fatty acids were branched-chain iso-C17:0 2-OH, iso-C15:0, iso-C17:1, and iso-C17:0. The strain showed the highest 16S rRNA gene sequence similarity to Corallococcusterminator CA054AT (99.67%) and C. praedator CA031BT (99.17%), and formed a novel branch both in the 16S rRNA gene sequence and phylogenomic tree. The genome size was 9,437,609 bp, with a DNA G + C content of 69.8 mol%. The strain had an average nucleotide identity (ANI) value lower than the species cut-off (95%), and with the digital DNA-DNA hybridization (dDDH) below the 70% threshold compared to the closest type strains. Secondary metabolite and biosynthetic gene cluster analyses revealed the strain's potential to produce novel compounds. Based on polyphasic taxonomic characterization, we propose that strain ZKHCc1 1396T represents a novel species, Corallococcus soli sp. nov. (NCCB 100659T = CIP 111634T).

Three New Stigmatellin Derivatives Reveal Biosynthetic Insights of Its Side Chain Decoration.

Myxobacteria generate natural products with unique chemical structures, which not only feature remarkable biological functions, but also demonstrate unprecedented biosynthetic assembly strategies. The stigmatellins have been previously described as potent inhibitors of the mitochondrial and photosynthetic respiratory chain and originate from an unusual polyketide synthase assembly line. While previous biosynthetic investigations were focused on the formation of the 5,7-dimethoxy-8-hydroxychromone ring, side chain decoration of the hydrophobic alkenyl chain in position 2 was investigated less thoroughly. We report here the full structure elucidation, as well as cytotoxic and antimicrobial activities of three new stigmatellins isolated from the myxobacterium Vitiosangium cumulatum MCy10943T with side chain decorations distinct from previously characterized members of this compound family. The hydrophobic alkenyl chain in position 2 of the herein described stigmatellins feature a terminal carboxylic acid group (1), a methoxy group at C-12' (2) or a vicinal diol (3). These findings provide further implications considering the side chain decoration of these aromatic myxobacterial polyketides and their underlying biosynthesis.

Myxobacteria of the Cystobacterineae Suborder Are Producers of New Vitamin K2 Derived Myxoquinones.

Vitamin K is an essential, lipid soluble vitamin that plays an important role in the human blood coagulation cascade as well as in the life cycle of bacteria and plants. In this study, we report the isolation and structure elucidation of unprecedented polyhydroxylated menaquinone variants named myxoquinones that are produced by myxobacteria and structurally belong to the Vitamin K family. We analyze the occurrence of myxoquinones across an LC-MS data collection from myxobacterial extracts and shed light on the distribution of myxoquinone-type biosynthetic gene clusters among publicly available myxobacterial genomes. Our findings indicate that myxoquinones are specifically produced by strains of the Cystobacterineae suborder within myxobacteria. Furthermore, bioinformatic analysis of the matching gene clusters allowed us to propose a biosynthetic model for myxoquinone formation. Due to their increased water-solubility, the myxoquinones could be a suitable starting point for the development of a better bioavailable treatment of vitamin K deficiency.

The Effects of Combined Respiratory-Gated Auricular Vagal Afferent Nerve Stimulation and Mindfulness Meditation for Chronic Low Back Pain: A Pilot Study.

OBJECTIVE: Respiratory-gated Auricular Vagal Afferent Nerve stimulation (RAVANS) is a safe nonpharmacological approach to managing chronic pain. The purpose of the current study was to examine (1) the feasibility and acceptability of RAVANS, combined with mindful meditation (MM) for chronic low back pain (CLBP), (2) the potential synergy of MM+RAVANS on improving pain, and (3) possible moderators of the influence of MM+RAVANS on pain. DESIGN: Pilot feasibility and acceptability study. SETTING: Pain management center at large academic medical center. SUBJECTS: Nineteen adults with CLBP and previous MM training. METHODS: Participants attended two sessions during which they completed quantitative sensory testing (QST), rated pain severity, and completed a MM+stimulation session. Participants received RAVANS during one visit and sham stimulation during the other, randomized in order. Following intervention, participants repeated QST. RESULTS: MM+RAVANS was well tolerated, acceptable, and feasible to provide relief for CLBP. Both MM+stimulation sessions resulted in improved back pain severity, punctate pain ratings, and pressure pain threshold. Individuals with greater negative affect showed greater back pain improvement from MM+RAVANS while those with greater mindfulness showed greater back pain improvement from MM+sham. CONCLUSIONS: Results suggest that for CLBP patients with prior MM training, the analgesic effects of MM may have overshadowed effects of RAVANS given the brief single session MM+RAVANS intervention. However, those with greater negative affect may benefit from combined MM+RAVANS.

New Deoxyenhygrolides from Plesiocystis pacifica Provide Insights into Butenolide Core Biosynthesis.

Marine myxobacteria present a virtually unexploited reservoir for the discovery of natural products with diverse biological functions and novel chemical scaffolds. We report here the isolation and structure elucidation of eight new deoxyenhygrolides (1-8) from the marine myxobacterium Plesiocystis pacifica DSM 14875T. The herein described deoxyenhygrolides C-J (1-8) feature a butenolide core with an ethyl residue at C-3 of the γ-lactone in contrast to the previously described derivatives, deoxyenhygrolides A and B, which feature an isobutyl residue at this position. The butenolide core is 2,4-substituted with a benzyl (1, 2 and 7), benzoyl (3 and 4) or benzyl alcohol (5, 6 and 8) moiety in the 2-position and a benzylidene (1-6) or benzylic hemiketal (7 and 8) in the 4-position. The description of these new deoxyenhygrolide derivatives, alongside genomic in silico investigation regarding putative biosynthetic genes, provides some new puzzle pieces on how this natural product class might be formed by marine myxobacteria.

Sandacrabins - Structurally Unique Antiviral RNA Polymerase Inhibitors from a Rare Myxobacterium.

Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

Structure and Biosynthesis of Myxofacyclines: Unique Myxobacterial Polyketides Featuring Varing and Rare Heterocycles[] *.

A metabolome-guided screening approach in the novel myxobacterium Corallococcus sp. MCy9072 resulted in the isolation of the unprecedented natural product myxofacycline A, which features a rare isoxazole substructure. Identification and genomic investigation of additional producers alongside targeted gene inactivation experiments and heterologous expression of the corresponding biosynthetic gene cluster in the host Myxococcus xanthus DK1622 confirmed a noncanonical megaenzyme complex as the biosynthetic origin of myxofacycline A. Induced expression of the respective genes led to significantly increased production titers enabling the identification of six further members of the myxofacycline natural product family. Whereas myxofacyclines A-D display an isoxazole substructure, intriguingly myxofacyclines E and F were found to contain 4-pyrimidinole, a heterocycle unprecedented in natural products. Lastly, myxofacycline G features another rare 1,2-dihydropyrol-3-one moiety. In addition to a full structure elucidation, we report the underlying biosynthetic machinery and present a rationale for the formation of all myxofacyclines. Unexpectedly, an extraordinary polyketide synthase-nonribosomal peptide synthetase hybrid was found to produce all three types of heterocycle in these natural products.

Respiratory-gated auricular vagal afferent nerve stimulation (RAVANS) modulates brain response to stress in major depression.

BACKGROUND: Negative stress significantly impacts major depressive disorder (MDD), given the shared brain circuitry between the stress response and mood. Thus, interventions that target this circuitry will have an important impact on MDD. The aim of this study was to evaluate the acute effects of a novel respiratory-gated auricular vagal afferent nerve stimulation (RAVANS) technique in the modulation of brain activity and connectivity in women with MDD in response to negative stressful stimuli. METHODS: Twenty premenopausal women with recurrent MDD in an active episode were included in a cross-over experimental study that included two functional MRI visits within one week, randomized to receive exhalatory- (e-RAVANS) or inhalatory-gated (i-RAVANS) at each visit. Subjects were exposed to a visual stress challenge that preceded and followed RAVANS. A Factorial analysis was used to evaluate the effects of RAVANS on brain activity and connectivity and changes in depressive and anxiety symptomatology post-stress. RESULTS: Compared with i-RAVANS, e-RAVANS was significantly associated with increased activation of subgenual anterior cingulate, orbitofrontal and ventromedial prefrontal cortices and increased connectivity between hypothalamus and dorsolateral prefrontal cortex, and from nucleus tractus solitarii to locus coeruleus and ventromedial prefrontal cortex. Changes in brain activity and connectivity after e-RAVANS were significantly associated with a reduction in depressive and anxiety symptoms. CONCLUSIONS: Our study suggests exhalatory-gated RAVANS effectively modulates brain circuitries regulating response to negative stress and is associated with significant acute reduction of depressive and anxiety symptomatology in women with recurrent MDD. Findings suggest a potential non-pharmacologic intervention for acute relief of depressive symptomatology in MDD.

Structure and biosynthesis of sorangipyranone - a new γ-dihydropyrone from the myxobacterial strain MSr12020.

Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength n→π* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study.