Janus kinase 2 inhibition by pacritinib as potential therapeutic target for liver fibrosis.

BACKGROUND AND AIMS: Janus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo . APPROACH AND RESULTS: Transcriptome analyses of JAK2 in human livers and other targets of pacritinib have been shown to correlate with profibrotic factors. Although transcription of JAK2 correlated significantly with type I collagen expression and other profibrotic genes, no correlation was observed for interleukin-1 receptor-associated kinase and colony-stimulating factor 1 receptor. Pacritinib decreased gene expression of fibrosis markers in mouse primary and human-derived HSCs in vitro . Moreover, pacritinib decreased the proliferation, contraction, and migration of HSCs. C 57 BL/6J mice received ethanol in drinking water (16%) or Western diet in combination with carbon tetrachloride intoxication for 7 weeks to induce alcoholic or nonalcoholic fatty liver disease. Pacritinib significantly reduced liver fibrosis assessed by gene expression and Sirius red staining, as well as HSC activation assessed by alpha-smooth muscle actin immunostaining in fibrotic mice. Furthermore, pacritinib decreased the gene expression of hepatic steatosis markers in experimental alcoholic liver disease. Additionally, pacritinib protected against liver injury as assessed by aminotransferase levels. CONCLUSIONS: This study demonstrates that the JAK2 inhibitor pacritinib may be promising for the treatment of alcoholic and nonalcoholic liver fibrosis and may be therefore relevant for human pathology.

Effects of Different Doses of sUV-B Exposure on Taxane Compounds' Metabolism in Taxus wallichiana var. Mairei.

UV-B is an important environmental factor that differentially affects plant growth and secondary metabolites. The effects of supplemental ultraviolet-B (sUV-B) exposure (T1, 1.40 kJ·m-2·day-1; T2, 2.81 kJ·m-2·day-1; and T3, 5.62 kJ·m-2·day-1) on the growth biomass, physiological characteristics, and secondary metabolites were studied. Our results indicated that leaf thickness was significantly (p < 0.05) reduced under T3 relative to the control (natural light exposure, CK); The contents of 6-BA and IAA were significantly reduced (p < 0.05); and the contents of ABA, 10-deacetylbaccatin III, and baccatin III were significantly (p < 0.05) increased under T1 and T2. The paclitaxel content was the highest (0.036 ± 0.0018 mg·g-1) under T3. The cephalomannine content was significantly increased under T1. Hmgr gene expression was upregulated under T1 and T3. The gene expressions of Bapt and Dbtnbt were significantly (p < 0.05) upregulated under sUV-B exposure, and the gene expressions of CoA, Ts, and Dbat were significantly (p < 0.05) downregulated. A correlation analysis showed that the 6-BA content had a significantly (p < 0.05) positive correlation with Dbat gene expression. The IAA content had a significantly (p < 0.05) positive correlation with the gene expression of Hmgr, CoA, Ts, and Dbtnbt. The ABA content had a significantly (p < 0.05) positive correlation with Bapt gene expression. Dbat gene expression had a significantly (p < 0.05) positive correlation with the 10-deacetylbaccatin content. Hmgr gene expression was positively correlated with the contents of baccatin III and cephalomannine. Bapt gene expression had a significantly (p < 0.01) positive correlation with the paclitaxel content. A factor analysis showed that the accumulation of paclitaxel content was promoted under T2, which was helpful in clarifying the accumulation of taxane compounds after sUV-B exposure.

Pyrrocidine, a molecular off switch for fumonisin biosynthesis.

Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides' protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.

Antimicrobial Mechanisms of Enterocin CHQS Against Candida albicans.

Candida albicans is the most common fungal pathogen in hospital-acquired infections, which is extremely harmful to health. The increasing fungal infections is requiring the rapid development of novel antifungal agents. In this study, the antimicrobial activity of CHQS, an enterocin isolated from Enterococcus faecalis TG2 against C. albicans was confirmed by the minimum inhibitory concentration, minimum fungicidal concentration, and time-kill curve. Aniline blue and calcofluor white staining methods showed that CHQS remarkably affected ß-1,3-glucan and chitin cell wall components and made cell wall more vulnerable. The C. albicans cell wall rupture and intracellular vacuolation were observed by TEM and SEM. Moreover, CHQS induced the accumulation of intracellular reactive oxygen species and decreased mitochondrial membrane potential. These results suggested that CHQS might have a complex multi-target antimicrobial mechanism against C. albicans. In addition, the use of CHQS combined with amphotericin B showed synergistic antimicrobial effects against C. albicans. In conclusion, enterocin CHQS, a natural product with antimicrobial effect, might has a bright future for the development of new antifungal drugs.

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α 2 ß 3 γ 2 GABAA Receptor.

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 ß 3 γ 2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 ß 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or ß 3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2ß3γ2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products.

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.

Reversible Control of DNA Binding with Cucurbit[8]uril-Induced Supramolecular 4,4'-Bipyridinium-Peptide Dimers.

Many cellular processes in living organisms are regulated by complex regulatory networks, built from noncovalent interactions between relatively few proteins that perform their functions by switching between homo- and heterooligomeric assemblies or mono- and bivalent states. Herein, we demonstrate that the conjugation of a 4,4'-bipyridinium scaffold to the basic region of the GCN4 bZip transcription factor can be exploited to control the dimerization of the conjugate by formation of a supramolecular complex with cucurbit[8]uril. Importantly, this supramolecular complex is able to specifically recognize its target dsDNA, and this binding can be reversibly switched by the application of external stimuli.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

AMOEBA binding free energies for the SAMPL7 TrimerTrip host-guest challenge.

As part of the SAMPL7 host-guest binding challenge, the AMOEBA force field was applied to calculate the absolute binding free energy for 16 charged organic ammonium guests to the TrimerTrip host, a recently reported acyclic cucurbituril-derived clip host structure with triptycene moieties at its termini. Here we report binding free energy calculations for this system using the AMOEBA polarizable atomic multipole force field and double annihilation free energy methodology. Conformational analysis of the host suggests three families of conformations that do not interconvert in solution on a time scale available to nanosecond molecular dynamics (MD) simulations. Two of these host conformers, referred to as the "indent" and "overlap" structures, are capable of binding guest molecules. As a result, the free energies of all 16 guests binding to both conformations were computed separately, and combined to produce values for comparison with experiment. Initial ranked results submitted as part of the SAMPL7 exercise had a mean unsigned error (MUE) from experimental binding data of 2.14 kcal/mol. Subsequently, a rigorous umbrella sampling reference calculation was used to better determine the free energy difference between unligated "indent" and "overlap" host conformations. Revised binding values for the 16 guests pegged to this umbrella sampling reference reduced the MUE to 1.41 kcal/mol, with a correlation coefficient (Pearson R) between calculated and experimental binding values of 0.832 and a rank correlation (Kendall τ) of 0.65. Overall, the AMOEBA results demonstrate no significant systematic error, suggesting the force field provides an accurate energetic description of the TrimerTrip host, and an appropriate balance of solvation and desolvation effects associated with guest binding.

SAMPL7 TrimerTrip host-guest binding poses and binding affinities from spherical-coordinates-biased simulations.

Host-guest binding remains a major challenge in modern computational modelling. The newest 7th statistical assessment of the modeling of proteins and ligands (SAMPL) challenge contains a new series of host-guest systems. The TrimerTrip host binds to 16 structurally diverse guests. Previously, we have successfully employed the spherical coordinates as the collective variables coupled with the enhanced sampling technique metadynamics to enhance the sampling of the binding/unbinding event, search for possible binding poses and calculate the binding affinities in all three host-guest binding cases of the 6th SAMPL challenge. In this work, we report a retrospective study on the TrimerTrip host-guest systems by employing the same protocol to investigate the TrimerTrip host in the SAMPL7 challenge. As no binding pose is provided by the SAMPL7 host, our simulations initiate from randomly selected configurations and are proceeded long enough to obtain converged free energy estimates and search for possible binding poses. The calculated binding affinities are in good agreement with the experimental reference, and the obtained binding poses serve as a nice starting point for end-point or alchemical free energy calculations. Note that as the work is performed after the close of the SAMPL7 challenge, we do not participate in the challenge and the results are not formally submitted to the SAMPL7 challenge.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

Isolation, Phylogenetic and Gephyromycin Metabolites Characterization of New Exopolysaccharides-Bearing Antarctic Actinobacterium from Feces of Emperor Penguin.

A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9-79.1%), average amino acid identity (AAI, 52.4-66.9%) and digital DNA-DNA hybridization (dDDH, 18.6-21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.

Site occupancy calibration of taxane pharmacology in live cells and tissues.

Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.

Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin.

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

A General Supramolecular Approach to Regulate Protein Functions by Cucurbit[7]uril and Unnatural Amino Acid Recognition.

Regulation of specific protein function is of great importance for both research and therapeutic development. Many small or large molecules have been developed to control specific protein function, but there is a lack of a universal approach to regulate the function of any given protein. We report a general host-guest molecular recognition approach involving modification of the protein functional surfaces with genetically encoded unnatural amino acids bearing guest side chains that can be specifically recognized by cucurbit[7]uril. Using two enzymes and a cytokine as models, we showed that the activity of proteins bearing unnatural amino acid could be turned off by host molecule binding, which blocked its functional binding surface. Protein activity can be switched back by treatment with a competitive guest molecule. Our approach provides a general tool for reversibly regulating protein function through molecular recognition and can be expected to be valuable for studying protein functions.

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.

Occurrence, synthesis and applications of natural and designed [3.3.3]propellanes.

Covering: 1978 to 2019 The synthetically challenging [3.3.3]propellane core has caught a lot of attention over the last 50 years. This comprehensive review details all synthetic strategies reported in the period 1978-2019 to facilitate the synthesis of carbocyclic [3.3.3]propellanes. The described strategies span from acid-catalyzed rearrangements and photo-mediated cycloadditions of ketones, heteropropellanes and dispiroundecanes to thermal rearrangements of acetylenes and alkenes. Other approaches, such as radical reactions with halogenated alkenes, domino cyclizations, the smart use of epoxide-carbonyl rearrangements and intramolecular palladium-catalyzed ring contractions are discussed as well. A special section is dedicated to triptindanes, a subclass of [3.3.3]propellanes which are of interest to material sciences.

Effects of temperature on larval development, alkaloid production and microsporidiosis in the two-spotted lady beetle, Adalia bipunctata L. (Coleoptera: Coccinellidae).

Nosema adaliae, a microsporidian pathogen described from the two-spotted lady beetle, Adalia bipunctata L., delays larval development when A. bipunctata is reared under laboratory conditions at 25 °C. In nature, lady beetles often experience a wide range of environmental temperatures, but little is known regarding the effects of microsporidian pathogens on lady beetles when they are reared at higher and lower temperatures. In this study, the effects of elevated rearing temperatures and microsporidiosis were observed on larval development time and mortality, sex ratios, alkaloid content (adaline and adalinine), and adult morphometrics. Uninfected larvae (24 h-old) were provided either an uninfected or N. adaliae-infected conspecific egg and subsequently reared at three temperatures (25 °C, 27.5 °C or 30 °C). After the egg was eaten, larvae were provided a diet of green peach aphids and their development was recorded daily. Following eclosion, a subsample of adults were photographed for microscopic measurements and reflex-fluid was collected for alkaloid analysis using gas chromatography-mass spectrometry. Afterwards, all individuals were examined to determine the number that had become infected, and the same subsample was used to assess the severity of infection. Development time was delayed significantly for larvae that consumed a N. adaliae-infected egg, and infected larvae took significantly longer to develop at 25 °C than at 27.5 °C or 30 °C. No differences were observed for larval mortality or sex ratios. The relative proportion of adaline increased when larvae were reared at 30 °C, but adaline concentration was not affected by the pathogen, N. adaliae. Adults reared at 25 °C had larger body measurements when compared to those reared at 27.5 °C and 30 °C. Overall, adults that had eaten a N. adaliae-infected A. bipunctata egg as larvae had similar body measurements to those that ate an uninfected egg. When comparing male and female measurements, a significant interaction between infection status, temperature and sex was observed for elytra length and head capsule width only. These measurements were similar for uninfected and N. adaliae-infected females across all temperatures. However, when reared at 25 °C, uninfected males had significantly smaller elytra and head capsules than did infected males; but when reared at 30 °C, no significant difference was observed for these measurements. Both percent infection and average spore count decreased at 27.5 °C and 30 °C. These results suggest that temperatures above 25 °C have a mitigating effect against N. adaliae in A. bipunctata.

Structural enterocin gene profiles and mode of antilisterial activity in synthetic liquid media and skim milk of autochthonous Enterococcus spp. isolates from artisan Greek Graviera and Galotyri cheeses.

The presence of eight common structural enterocin genes, singly or in varying combinations, in the genome of 15 antagonistic Enterococcus spp. previously isolated from artisan Greek Graviera and Galotyri retail cheeses was tested and associated with the mode of enterocin (Ent+) antilisterial activity of each isolate in three liquid culture media. The isolates were assigned to nine distinct strain genotypes of E. faecium (4 strains), E. durans (2) and E. faecalis (3). All strains were non-hemolytic, except for a cyl-positive E. faecalis genotype isolated from Galotyri cheese, which was strongly listericidal. All other strains varied from being listeriostatic to weakly listericidal in MRS and M17 broth, whereas all failed to inhibit listerial growth in skim milk. Two E. faecium strains retained strong Ent+ activity following neutralization and filter-sterilization of their MRS or M17 co-culture supernatants, whereas, all others required contact or proximity of their viable cells with L. monocytogenes cells in order to display activity. Additional studies to evaluate safety and potential synergistic effects of each strain genotype with starter LAB species in real milk environments will reveal the most active and truly harmless Enterococcus genotypes to be applied as co-starter or bioprotective adjunct cultures in traditional Greek cheese technologies.