Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Engineered phage endolysin eliminates Gardnerella in bacterial vaginosis without damaging the healthy vaginal microbiome

10.1101/2020.10.21.20216853 ◽

2020 ◽

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Agnieszka Latka ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Ex Vivo ◽

Bacterial Species ◽

Drug Candidate ◽

Vaginal Microbiome ◽

Promising Alternative ◽

Binding Domains ◽

Cell Wall Binding ◽

Homologous Genes

BackgroundBacterial vaginosis is characterized by an imbalance of the vaginal microbiome in which the normally predominant lactobacilli are replaced by other bacterial species. Initiated by strains of the bacterium Gardnerella, a characteristic biofilm forms on the vaginal epithelium, explaining the typical presence of clue cells. This biofilm contributes to the resilience of the bacteria to antibiotic treatment, which may explain the frequent recurrence of BV.ObjectiveIn this study, we investigate whether a therapy based on bacteriophage endolysins which specifically lyse Gardnerella, in particular the drug candidate PM-477, might be a promising alternative to broad-spectrum antibiotics and antiseptics.Study designTo identify Gardnerella specific endolysins, we searched for endolysin-encoding sequences in regions of Gardnerella genomes that are of prophage origin. This search identified fourteen homologous genes predicted to encode 1,4-beta-N-acetylmuramidase-type endolysins. When expressed in Escherichia coli and purified, the recombinant proteins demonstrated strong bactericidal activity against four different Gardnerella species. By shuffling the N-terminal catalytic domains and C-terminal cell wall-binding domains between the homologues, we produced 81 chimeric endolysins. These endolysins were tested for their activity and specificity in vitro and ex-vivo on vaginal samples from fifteen BV positive patients. Fluorescence in situ hybridization was used for visualization.ResultsSeveral engineered endolysins were 10-fold more active than the most active wild-type enzymes. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/ml. PM-477 had no effect on Lactobacillus strains or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested on vaginal samples from fifteen patients with either first time or recurring bacterial vaginosis. In fourteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome.ConclusionThe high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising drug candidate and an alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.10.137 ◽

2014 ◽

Vol 10 ◽

pp. 1347-1353 ◽

Cited By ~ 9

Author(s):

Anna M Bogazkaya ◽

Clemens J von Bühler ◽

Sebastian Kriening ◽

Alexandrine Busch ◽

Alexander Seifert ◽

...

Keyword(s):

High Selectivity ◽

Allylic Alcohols ◽

Thermobifida Fusca ◽

Wild Type ◽

Cytochrome P450 Enzymes ◽

Wild Type Enzyme ◽

Total Product ◽

Regioselective Oxidation ◽

Allylic Hydroxylation ◽

Enzyme Selectivity

Allylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC–MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.

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Hydrogenolysis cleavage of Csp2-Csp3 bond over a metal-free NbOPO4 catalyst

10.26434/chemrxiv-2021-g3gbd ◽

2021 ◽

Author(s):

Hao Zhou ◽

Lu Chen ◽

Yong Guo ◽

Xiaohui Liu ◽

Xin-Ping Wu ◽

...

Keyword(s):

Density Functional ◽

Oxygen Vacancies ◽

High Selectivity ◽

Bond Cleavage ◽

Acid Sites ◽

Model Compounds ◽

Functional Theory ◽

In Situ Drift ◽

First Time

Ru/NbOx catalysts, which combine the merits of facile hydrogen activation, strong binding to benzene ring and the presence of Brønsted acid sites, were well investigated toward Csp2-Csp3 bond cleavage. Herein, we unlock the ability of bare NbOx catalyst in the dissociation and activation of hydrogen molecule and further hydrogenolysis of the Csp2-Csp3 model compounds including polystyrene (PS). In-situ Drift and density functional theory (DFT) calculations reveal that H2 can be dissociated and surface hydride species can be produced over Nb2O5 through heterolytic and homolytic cleavages of H2. We also find that the existence of surface oxygen vacancies plays a key role in stabilizing hydride species. Further, the NbOPO4 catalyst not only allows the conversion of phenylcyclohexane to monocyclic compounds by cleaving Csp2-Csp3 bond, but also enables the conversion of PS to arenes with a high selectivity. This study provides and proves for the first time, the unique ability of metal oxides (phosphates) in the hydrogenolysis of compounds and plastics containing Csp2-Csp3 bonds.

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Low Prevalence of Pfcrt Resistance Alleles among Patients with Uncomplicated Falciparum Malaria in Niger Six Years after Chloroquine Withdrawal

Malaria Research and Treatment ◽

10.1155/2014/614190 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7

Author(s):

Adamou Salissou ◽

Halima Zamanka ◽

Brigitte Biyghe Binze ◽

Taiana Rivière ◽

Magalie Tichit ◽

...

Keyword(s):

Public Health ◽

Sequence Data ◽

Ex Vivo ◽

Significant Decline ◽

Wild Type ◽

Blood Samples ◽

Few Data ◽

Low Prevalence ◽

First Time ◽

Resistant Genotypes

Chloroquine (CQ) resistance is widespread in Africa, but few data are available for Niger. Pfcrt haplotypes (aa 56–118) and ex vivo responses to CQ and amodiaquine were characterized for 26 isolates collected in South Niger from children under 15 years of age suffering from uncomplicated falciparum malaria, six years after the introduction of artemisinin-based combinations and the withdrawal of CQ. The wild-type Pfcrt haplotype CVMNK was found in 22 of the 26 isolates, with CVIET sequences observed in only three of the samples. We also describe for the first time a new CVINT haplotype. The ex vivo responses were better for CVMNK than for CVIET parasites. Pfcrt sequence data were compared with those obtained for 26 additional parasitized blood samples collected in Gabon, from an area of CQ resistance used as a control. Our findings suggest that there has been a significant decline in CQ-resistant genotypes since the previous estimates for Niger were obtained. No such decline in molecular resistance to CQ was observed in the subset of samples collected in similar conditions from Gabon. These results have important implications for public health and support the policy implemented in Niger since 2005, which aims to increase the efficacy and availability of antimalarial drugs whilst controlling the spread of resistance.

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Comparison Between Conventional and Supersaturable Self-nanoemulsion Loaded with Nebivolol: Preparation and In-vitro/Ex-vivo Evaluation

Iraqi Journal of Pharmaceutical Sciences ( P-ISSN 1683 - 3597 E-ISSN 2521 - 3512) ◽

10.31351/vol29iss1pp216-225 ◽

2020 ◽

Vol 29 (1) ◽

pp. 216-225

Author(s):

Lubna A. Sabri ◽

Ahmed A. Hussein

Keyword(s):

High Selectivity ◽

Ex Vivo ◽

Permeation Rate ◽

Promising Alternative ◽

Precipitation Inhibition ◽

Ex Vivo Permeation ◽

Alternative Approach ◽

Polymer Type

Nebivolol (NBH) is a third-generation B1-blocker with high selectivity and vasodilation activity. Nevertheless, nebivolol exhibits low oral bioavailability, which may adversely affect its efficacy. Recently, supersaturable self-nanoemulsion (Su-SNE) is an advanced SNE approach that can address low bioavailability The study aims to prepare nebivolol-loaded Su-SNE by reduction the amount of the prepared conventional SNE to half. Besides, an appropriate polymer type and concentration to prevent NBH precipitation upon oral administration have investigated.. A conventional self-nanoemulsion (formula A) was prepared by dissolving NBH in 500 mg vehicle mixture of imwitor®988: cremophor-EL: propylene glycol. Then, eight Su-SNE formulas with the absence or presence of four different polymers were prepared and evaluated. In-vitro precipitation assay was performed to assess the precipitation inhibition capacity of polymers. The ex-vivo permeation through rat intestinal mucosa was also conducted for determination of permeability parameters. Results revealed that (Su-SNA formula SAS1) containing 5% soluplus could effectively retard the nebivolol precipitation. There was no statistical difference between formula A and SAS1; both maintained a higher apparent NBH concentration for approximately 240 min in 0.1N HCl. The permeation rate of conventional (formula A) and soluplus-based Su-SNE (formula SAS1) was significantly improved, and the permeation enhancement ratio was found 2.7 and 3.2, respectively, as compared with non-formulated NBH. Consequently, it is concluded that developing soluplus-based nebivolol SNE is a promising alternative approach. It can enhance nebivolol stability and permeability with half the amount of conventional SNE components.

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Probing the Dynamic Self-Assembly Behaviour of Photoswitchable Wormlike Micelles in Real Time

10.26434/chemrxiv.7098695 ◽

2018 ◽

Author(s):

Elaine A. Kelly ◽

Judith E. Houston ◽

Rachel Evans

Keyword(s):

Real Time ◽

Absorption Spectroscopy ◽

Self Assembly ◽

Uv Light ◽

Wormlike Micelles ◽

Tetraethylene Glycol ◽

Light Illumination ◽

First Time ◽

Dependent Processes

Understanding the dynamic self-assembly behaviour of azobenzene photosurfactants (AzoPS) is crucial to advance their use in controlled release applications such asdrug delivery and micellar catalysis. Currently, their behaviour in the equilibrium cis-and trans-photostationary states is more widely understood than during the photoisomerisation process itself. Here, we investigate the time-dependent self-assembly of the different photoisomers of a model neutral AzoPS, <a>tetraethylene glycol mono(4′,4-octyloxy,octyl-azobenzene) </a>(C8AzoOC8E4) using small-angle neutron scattering (SANS). We show that the incorporation of in-situUV-Vis absorption spectroscopy with SANS allows the scattering profile, and hence micelle shape, to be correlated with the extent of photoisomerisation in real-time. It was observed that C8AzoOC8E4could switch between wormlike micelles (transnative state) and fractal aggregates (under UV light), with changes in the self-assembled structure arising concurrently with changes in the absorption spectrum. Wormlike micelles could be recovered within 60 seconds of blue light illumination. To the best of our knowledge, this is the first time the degree of AzoPS photoisomerisation has been tracked in-situthrough combined UV-Vis absorption spectroscopy-SANS measurements. This technique could be widely used to gain mechanistic and kinetic insights into light-dependent processes that are reliant on self-assembly.

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The Total Synthesis of Rhabdastrellic Acid A

10.26434/chemrxiv.7479944 ◽

2018 ◽

Author(s):

Yaroslav Boyko ◽

Christopher Huck ◽

David Sarlah

Keyword(s):

Total Synthesis ◽

Late Stage ◽

Cross Coupling ◽

Side Chains ◽

General Strategy ◽

Modular Approach ◽

Linear Sequence ◽

Generating Sequence ◽

First Time

<div>The first total synthesis of rhabdastrellic acid A, a highly cytotoxic isomalabaricane triterpenoid, has been accomplished in a linear sequence of 14 steps from commercial geranylacetone. The prominently strained trans-syn-trans-perhydrobenz[e]indene core characteristic of the isomalabaricanes is efficiently accessed in a selective manner for the first time through a rapid, complexity-generating sequence incorporating a reductive radical polyene cyclization, an unprecedented oxidative Rautenstrauch cycloisomerization, and umpolung 𝛼-substitution of a p-toluenesulfonylhydrazone with in situ reductive transposition. A late-stage cross-coupling in concert with a modular approach to polyunsaturated side chains renders this a general strategy for the synthesis of numerous family members of these synthetically challenging and hitherto inaccessible marine triterpenoids.</div>

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