Augmented KCa2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice

Uterine contractions prior to 37 weeks gestation can result in preterm labor with significant risk to the infant. Current tocolytic therapies aimed at suppressing premature uterine contractions are largely ineffective and cause serious side effects. Calcium (Ca2+) dependent contractions of uterine smooth muscle are physiologically limited by the opening of membrane potassium (K+) channels. Exploiting such inherent negative feedback mechanisms may offer new strategies to delay labor and reduce risk. Positive modulation of small conductance Ca2+-activated K+ (KCa2.3) channels with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), effectively decreases uterine contractions. This study investigates whether the receptor agonist oxytocin might solicit KCa2.3 channel feedback that facilitates CyPPA suppression of uterine contractions. Using isometric force myography, we found that spontaneous phasic contractions of myometrial tissue from nonpregnant mice were suppressed by CyPPA and, in the presence of CyPPA, oxytocin failed to augment contractions. In tissues exposed to oxytocin, depletion of internal Ca2+ stores with cyclopiazonic acid (CPA) impaired CyPPA relaxation, whereas blockade of nonselective cation channels (NSCC) using gadolinium (Gd3+) had no significant effect. Immunofluorescence revealed close proximity of KCa2.3 channels and ER inositol trisphosphate receptors (IP3Rs) within myometrial smooth muscle cells. The findings suggest internal Ca2+ stores play a role in KCa2.3-dependent feedback control of uterine contraction and offer new insights for tocolytic therapies.

The Solvent Dimethyl Sulfoxide Affects Physiology, Transcriptome and Secondary Metabolism of Aspergillus flavus

Dimethyl sulfoxide (DSMO) is a simple molecule widely used because of its great solvating ability, but this solvent also has little-known biological effects, especially on fungi. Aspergillus flavus is a notorious pathogenic fungus which may contaminate a large variety of crops worldwide by producing aflatoxins, endangering at the same time food safety and international trade. The aim of this study was to characterize the effect of DMSO on A. flavus including developmental parameters such as germination and sporulation, as well as its transcriptome profile using high-throughput RNA-sequencing assay and its impact on secondary metabolism (SM). After DMSO exposure, A. flavus displayed depigmented conidia in a dose-dependent manner. The four-day exposition of cultures to two doses of DMSO, chosen on the basis of depigmentation intensity (35 mM “low” and 282 mM “high”), led to no significant impact on fungal growth, germination or sporulation. However, transcriptomic data analysis showed that 4891 genes were differentially regulated in response to DMSO (46% of studied transcripts). A total of 4650 genes were specifically regulated in response to the highest dose of DMSO, while only 19 genes were modulated upon exposure to the lowest dose. Secondary metabolites clusters genes were widely affected by the DMSO, with 91% of clusters impacted at the highest dose. Among these, aflatoxins, cyclopiazonic acid and ustiloxin B clusters were totally under-expressed. The genes belonging to the AFB1 cluster were the most negatively modulated ones, the two doses leading to 63% and 100% inhibition of the AFB1 production, respectively. The SM analysis also showed the disappearance of ustiloxin B and a 10-fold reduction of cyclopiazonic acid level when A. flavus was treated by the higher DMSO dose. In conclusion, the present study showed that DMSO impacted widely A. flavus’ transcriptome, including secondary metabolism gene clusters with the aflatoxins at the head of down-regulated ones. The solvent also inhibits conidial pigmentation, which could illustrate common regulatory mechanisms between aflatoxins and fungal pigment pathways. Because of its effect on major metabolites synthesis, DMSO should not be used as solvent especially in studies testing anti-aflatoxinogenic compounds.

Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50–3 days 4.50, 18.82, and 60.51 μg mL–1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations > 25 μg mL–1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett–Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 μg mL–1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.

Mycobiota and Mycotoxin Contamination of Traditional and Industrial Dry-Fermented Sausage Kulen

The aim of this study was to identify and compare surface mycobiota of traditional and industrial Croatian dry-fermented sausage Kulen, especially toxicogenic species, and to detect contamination with mycotoxins recognized as the most important for meat products. Identification of mould species was performed by sequence analysis of beta- tubulin and calmodulin gene, while the determination of mycotoxins aflatoxin B1 (AFB1), ochratoxin A (OTA), and cyclopiazonic acid (CPA) was carried out using the LC-MS/MS (liquid chromatography-tandem mass spectrometry) method. The results showed a significantly higher number of mould isolates and greater species (including of those mycotoxigenic) diversity in traditional Kulen samples in comparison with the industrial ones. P. commune, as a potential CPA-producer, was the most represented in traditional Kulen (19.0%), followed by P. solitum (16.6%), which was the most represented in industrial Kulen samples (23.8%). The results also showed that 69% of the traditional sausage samples were contaminated with either CPA or OTA in concentrations of up to 13.35 µg/kg and 6.95 µg/kg, respectively, while in the industrial samples only OTA was detected (in a single sample in the concentration of 0.42 µg/kg). Mycotoxin AFB1 and its producers were not detected in any of the analysed samples (<LOD).

Muscle stretching induces twitch contractions without activation of stretch-activated channels in intact rat trabeculae

Introduction Mechano-electric coupling (MEC) means that muscle stretching can induce action potentials. Stretch-activated channels (SACs) have been believed to play important roles in their induction. Purpose To investigate what degree of muscle stretching can induce MEC-mediated action potentials and what roles SACs play in their induction. Methods Trabeculae were obtained from right ventricles of rat hearts. Force was measured with a strain gauge, sarcomere length (SL) with a laser diffraction technique, and [Ca2+]i with fura-2 (24°C). The SL was set at 2.0 μm at the resting condition. Trabeculae were stimulated electrically at 400-ms intervals for 7.5 s. Various degrees of muscle stretching were applied at 500 ms after the last stimulus of the electrical train to determine the minimal SL (SL-AP) at which an action potential or a twitch contraction was induced by the stretching (0.7 mM [Ca2+]o). Results The SL-AP was 2.34±0.02 μm (n=8) when trabeculae were stretched rapidly from a SL of 2.0 μm (400-ms stimulation intervals, 0.7 mM [Ca2+]o). The SL-AP was not changed by increasing the stimulation intervals from 400 to 2000 ms (n=7), by increasing [Ca2+]o from 0.7 to 2 mM (n=8), and by adding 1 μM isoproterenol (n=8), suggesting that Ca2+ loading within the myocardium has no effect on the SL-AP. Surprisingly, the SL-AP was not changed by adding 5 μM GsMTx4 (n=8), 10 mM Gd3+ (n=9), 100 μM (n=8) and 200 μM streptomycin (n=11), revealing that SACs play no roles in the determination of SL-AP. The SL-AP was not changed by adding 1 μM ryanodine (n=5) and 30 μM cyclopiazonic acid and was not changed by adding 3 μM diphenyleneiodonium chloride (n=8) and 10 μM colchicine, suggesting that Ca2+ leak from the SR and activation of NADPH oxidase has no effect on the SL-AP. In contrast, elevation of temperature from 23 to 36°C decreased the SL-AP from 2.35±0.01 to 2.34±0.02 μm (p&lt;0.05, n=7). Elevation of extracellular K+ ([K+]o) from 5 to 10 mM increased the SL-AP from 2.35±0.01 to 2.38±0.01 μm (p&lt;0.01, n=7), while reduction of [K+]o to 5 mM decreased it to 2.36±0.01 μm (p&lt;0.05, n=7), suggesting that depolarization of membrane potential suppresses MEC-mediated twitch contractions. The SL-AP was increased from 2.34±0.01 to 2.36±0.01 μm (p&lt;0.01, n=7) when stretching was applied at a shorter interval after the last stimulus, i.e., 200 ms. After electrical stimulation at 300-ms stimulation intervals for 30 s, arrhythmias were induced by a MEC-mediated twitch contraction in 6 out of 9 trabeculae when stretching was applied at 500 ms after the last stimulus, while they were induced only in 2 out of 9 trabeculae without the stretching (4 mM [Ca2+]o, 1 μM isoproterenol). Conclusions These results suggest that muscle stretching causes membrane excitation, which potentially induces arrhythmias and that activation of SACs, Ca2+ release from the SR, and activation of NADPH oxidase by muscle stretching are not involved in the excitation. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science.

Mass Spectrometry-Based Network Analysis Reveals New Insights Into the Chemodiversity of 28 Species in Aspergillus section Flavi

Aspergillus section Flavi includes some of the most famous mycotoxin producing filamentous fungi known to mankind. In recent years a number of new species have been included in section Flavi, however these species have been much less studied from a chemical point of view. In this study, we explored one representative strain of a total of 28 fungal species in section Flavi by systematically evaluating the relationship between taxonomy and secondary metabolites with LC-MS/MS analysis for the first time and dereplication through an in-house database and the Global Natural Product Social Molecular Networking (GNPS) platform. This approach allowed rapid identification of two new cyclopiazonic acid producers (A. alliaceus and A. arachidicola) and two new tenuazonic acid producers (A. arachidicola and A. leporis). Moreover, for the first time we report species from section Flavi to produce fumifungin and sphingofungins B-D. Altogether, this study emphasizes that the chemical diversity of species in genus Aspergillus section Flavi is larger than previously recognized, and especially that understudied species are prolific producers of important mycotoxins such as fumi- and sphingofungins not previously reported from this section. Furthermore, our work demonstrates Global Natural Product Social (GNPS) Molecular Networking as a powerful tool for large-scale chemotaxonomic analysis of closely related species in filamentous fungi.

Testing the extraction of 12 mycotoxins from aqueous solutions by insoluble beta-cyclodextrin bead polymer

Mycotoxins are toxic metabolites of filamentous fungi; they are common contaminants in numerous foods and beverages. Cyclodextrins are ring-shaped oligosaccharides, which can form host-guest type complexes with certain mycotoxins. Insoluble beta-cyclodextrin bead polymer (BBP) extracted successfully some mycotoxins (e.g., alternariol and zearalenone) from aqueous solutions, including beverages. Therefore, in this study, we aimed to examine the ability of BBP to remove other 12 mycotoxins (including aflatoxin B1, aflatoxin M1, citrinin, dihydrocitrinone, cyclopiazonic acid, deoxynivalenol, ochratoxin A, patulin, sterigmatocystin, zearalanone, α-zearalanol, and β-zearalanol) from different buffers (pH 3.0, 5.0, and 7.0). Our results showed that BBP can effectively extract citrinin, dihydrocitrinone, sterigmatocystin, zearalanone, α-zearalanol, and β-zearalanol at each pH tested. However, for the removal of ochratoxin A, BBP was far the most effective at pH 3.0. Based on these observations, BBP may be a suitable mycotoxin binder to extract certain mycotoxins from aqueous solutions for decontamination and/or for analytical purposes.

Evaluation of Negative Inotropic Effects of a Isoquinoline Alkaloid N-14

In studies, the alkaloid 1-(2-Chloro-4,5-methylenedioxyphenyl)-2-hydroxyethyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (N-14) had a negative inotropic effect on the activity of the papillary muscle contraction of the rat heart detected. Ca2+ ions from SR play an important role in the process of contraction of the heart muscle. With this in mind, the negative inotropic effect of the N-14 alkaloid was investigated with the modification of the accumulation processes of Ca2+ ions to SR. To clarify this, we examined the effects of the alkaloid being studied on SERCA2a and RyR2. To do this, the inhibitor of SERCA2a - cyclopiazonic acid (CPA) and RyR activator caffeine, which provide the accumulation of Ca2+ ions in SR, were used.

Biological Activity of Extracts from Aromatic Plants as Control Agents against Spoilage Molds Isolated from Sheep Cheese

The aim of this work was to assess the antifungal and antioxidant activity of essential oils and ethanolic extracts from distilled solid by-products from aromatic plants (Artemisia dracunculus, Hyssopus officinalis, Lavandula stoechas, Origanum vulgare and Satureja montana) against 14 fungi strains isolated from sheep cheese and identified at species level using DNA barcoding based on β-tubulin sequence analysis. In addition, capacity of fungi to produce ochratoxin A, patulin, cyclopiazonic acid and sterigmatocystin was analyzed. Of the isolates, 85.7% belonged to Penicillium (P. commune/biforme, P. crustosum) and 14.3% to Aspergillus (A. puulaauensis and A. jensenii), the first time that these Aspergillus species have been found in sheep’s cheese. All P. commune isolates were producers of cyclopiazonic acid, and the two Aspergillus strains produced sterigmatocystin, but the others did not produce any tested mycotoxin. Among the essential oils tested, oregano, savory and tarragon had a significant antifungal activity against all the isolated strains, but no ethanolic extract showed antifungal activity. By contrast, ethanolic extracts showed great potential as antioxidants. The identification of new molds in cheese will help the dairy industry to know more about those molds affecting the sector, and the use of aromatic plants in the control of fungal spoilage could be a suitable alternative to chemical preservatives used in the agri-food industry.