Response of the mushroom pathogen Cladobotryum mycophilum to prochloraz and metrafenone fungicides and Streptomyces flavovirens actinobacteria.

After an outbreak of cobweb disease of cultivated button mushroom in Serbia in 2003, the isolated fungal pathogen was initially identified as Cladobotryum dendroides (teleomorph Hypomyces rosellus) based on morpho-physiological traits. Molecular analysis indicated re-classification of two strains (isolated in 2004 and 2007) as Cladobotryum mycophilum (teleomorph Hypomyces odoratus). However, subsequent analysis of further five strains (isolated over the period 2003-2010) within the frames of the present study, also confirmed their identification as the exclusive cobweb causal agent C. mycophilum. After artificial inoculation, the symptoms observed on harvested and growing mushrooms were consistent with the appearance of cobweb disease. Pathogen sensitivity to fungicides was estimated by probit analyses. Fungicide susceptibility tests showed that C. mycophilum strains were highly sensitive both to prochloraz (ED50<0.087 µg mL-1) and the newly introduced metrafenone (ED50<0.15 µg mL-1). Furthermore, the growth of all examined strains of C. mycophilum was significantly inhibited by the indigenous actinobacterial strain Streptomyces flavovirens A06. A dual culture assay showed after 72 h that the percentage of radial growth inhibition of the pathogen ranged from 22.38 to 55.73%. Our findings suggest that the antagonistic S. flavovirens A06 might be a potential candidate for controlling the cobweb disease of cultivated button mushroom.

Diversity and Content of Carotenoids and Other Pigments in the Transition from the Green to the Red Stage of Haematococcus pluvialis Microalgae Identified by HPLC-DAD and LC-QTOF-MS.

H. pluvialis is a unicellular freshwater alga containing many bioactive compounds, especially carotenoids, which are the strongest antioxidants among the pigments. This study evaluates the composition and content of carotenoids and other pigments in both stages of algae life cycle, especially in the green vegetative stage, less studied in comparison to the red stage. To determine the composition and content of carotenoids, a combination of HPLC-DAD and LC-QTOF-MS was used. The content of carotenoids in the green vegetative stage was significantly lower than in the red vegetative stage. In the green vegetative stage, 16 different carotenoids and other pigments were identified. Among the total 8.86 mg g-1 DW of pigments, 5.24 mg g-1 DW or 59% of them were chlorophyll a with its derivatives, and 3.62 mg g-1 DW or 41% of them were free carotenoids. After the transition from the green to the red stage, the carotenoid composition was replaced by secondary carotenoids, astaxanthin and its esters, which predominated in the whole carotenoid composition. In addition to free astaxanthin, 12 astaxanthin monoesters, 6 diesters and 13 other carotenoids were determined. The majority of 37.86 mg g-1 DW pigments were monoesters. They represented 82% of all pigments, and their content was about 5 times higher than both, diesters (5.91 mg g-1 DW or 12% of all) and free carotenoids (2.4 mg g-1 DW or 6% of all). The results of the study contribute to the data on the overall pigment composition and content of H. pluvialis algae and provide the basis for further improvement of cultivation of the H. pluvialis algae.

Identification and Content of Astaxanthin and Its Esters from Microalgae Haematococcus pluvialis by HPLC-DAD and LC-QTOF-MS after Extraction with Various Solvents.

Haematococcus pluvialis, a unicellular green microalga that produces a secondary metabolite under stress conditions, bears one of the most potent antioxidants, namely xanthophyll astaxanthin. The aim of our study was to determine the content of astaxanthin and its esterified forms using three different solvents-methyl tert-butyl ether (MTBE), hexane isopropanol (HEX -IPA) and acetone (ACE)-and to identify them by using high performance liquid chromatography coupled with diode array detection and the quadrupole time-of-flight mass spectrometry (HPLC-DAD and LC-QTOF-MS) technique. We identified eleven astaxanthin monoesters, which accounted for 78.8% of the total astaxanthin pool, six astaxanthin diesters (20.5% of total), while free astaxanthin represented the smallest fraction (0.7%). Astaxanthin monoesters (C16:2, C16:1, C16:0), which were the major bioactive compounds in the H. pluvialis samples studied, ranged from 10.2 to 11.8 mg g-1 DW. Astaxanthin diesters (C18:4/C18:3, C18:1/C18:3) were detected in the range between 2.3 and 2.6 mg g-1 DW. All three solvents were found to be effective for extraction, but MTBE and hexane-isopropanol extracted the greatest amount of free bioactive astaxanthin. Furthermore, MTBE extracted more low-chain astaxanthin monoesters (C16), and hexane-isopropanol extracted more long-chain monoesters (C18 and above) and more diesters. We can conclude that MTBE is the solvent of choice for the extraction of monoesters and hexane-isopropanol for diesters.

The freeze-all strategy versus agonist triggering with low-dose hCG for luteal phase support in IVF/ICSI for high responders: a randomized controlled trial.

STUDY QUESTION: Does the freeze-all strategy in high-responders increase pregnancy rates and improve safety outcomes when compared with GnRH agonist triggering followed by low-dose hCG intensified luteal support with a fresh embryo transfer? SUMMARY ANSWER: Pregnancy rates after either fresh embryo transfer with intensified luteal phase support using low-dose hCG or the freeze-all strategy did not vary significantly; however, moderate-to-severe ovarian hyperstimulation syndrome (OHSS) occurred more frequently in the women who attempted a fresh embryo transfer. WHAT IS KNOWN ALREADY: Two strategies following GnRH agonist triggering (the freeze-all approach and a fresh embryo transfer attempt using a low-dose of hCG for intensified luteal phase support) are safer alternatives when compared with conventional hCG triggering with similar pregnancy outcomes. However, these two strategies have never been compared head-to-head in an unrestricted predicted hyper-responder population. STUDY DESIGN, SIZE, DURATION: This study included women with an excessive response to ovarian stimulation (≥18 follicles measuring ≥11 mm) undergoing IVF/ICSI in a GnRH antagonist suppressed cycle between 2014 and 2017. Our primary outcome was clinical pregnancy at 7 weeks after the first embryo transfer. Secondary outcomes included live birth and the development of moderate-to-severe OHSS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Following GnRH agonist triggering, women were randomized either to cryopreserve all good-quality embryos followed by a frozen embryo transfer in an subsequent artificial cycle or to perform a fresh embryo transfer with intensified luteal phase support (1500 IU hCG on the day of oocyte retrieval, plus oral estradiol 2 mg two times a day, plus 200 mg of micronized vaginal progesterone three times a day). MAIN RESULTS AND THE ROLE OF CHANCE: A total of 212 patients (106 in each arm) were recruited in the study, with three patients (one in the fresh embryo transfer group and two in the freeze-all group) later withdrawing their consent to participate in the study. One patient in the freeze-all group became pregnant naturally (clinical pregnancy diagnosed 38 days after randomization) prior to the first frozen embryo transfer. The study arms did not vary significantly in terms of the number of oocytes retrieved and embryos produced/transferred. The intention to treat clinical pregnancy and live birth rates (with the latter excluding four cases lost to follow-up: one in the fresh transfer and three in the freeze-all arms, respectively) after the first embryo transfer did not vary significantly among the fresh embryo transfer and freeze-all study arms: 51/105 (48.6%) versus 57/104 (54.8%) and 41/104 (39.4%) versus 42/101 (41.6%), respectively (relative risk for clinical pregnancy 1.13, 95% CI 0.87-1.47; P = 0.41). However, moderate-to-severe OHSS occurred solely in the group that received low-dose hCG (9/105, 8.6%, 95% CI 3.2% to 13.9% vs 0/104, 95% CI 0 to 3.7, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The sample size calculation was based on a 19% absolute difference in terms of clinical pregnancy rates, therefore smaller differences, as observed in the trial, cannot be reliably excluded as non-significant. WIDER IMPLICATIONS OF THE FINDINGS: This study offers the first comparative analysis of two common strategies applied to women performing IVF/ICSI with a high risk to develop OHSS. While pregnancy rates did not vary significantly, a fresh embryo transfer with intensified luteal phase support may still not avoid the risk of moderate-to-severe OHSS and serious consideration should be made before recommending it as a routine first-line treatment. Future trials may allow us to confirm these findings. STUDY FUNDING/COMPETING INTEREST(S): The authors have no conflicts of interest to disclose. No external funding was obtained for this study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier NCT02148393. TRIAL REGISTRATION DATE: 28 May 2014. DATE OF FIRST PATIENT'S ENROLMENT: 30 May 2014.

Is ovarian response associated with adverse perinatal outcomes in GnRH antagonist IVF/ICSI cycles?

RESEARCH QUESTION: Is there an association between ovarian response and perinatal outcomes? DESIGN: A retrospective, single-centre cohort study including all women undergoing their first ovarian stimulation cycle in a gonadotrophin releasing hormone antagonist protocol, with a fresh embryo transfer that resulted in a singleton live birth from January 2009 to December 2015. Patients were categorized into four groups according to the number of oocytes retrieved: one to three (category 1), four to nine (category 2), 10-15 (category 3), or over 15 oocytes (category 4). RESULTS: The overall number of patients analysed was 964. No relevant statistical difference was found among neonatal outcomes across the four ovarian response categories. Neonatal weight (in grams) was comparable between all groups (3222 ± 607 versus 3254 ± 537 versus 3235 ± 575 versus 3200 ± 622; P = 0.85, in categories 1, 2, 3 and 4, respectively). No statistically significant differences were found among the ovarian response categories for birth weight z-scores (taking into account neonatal sex and delivery term). The incidence of pre-term birth and low birth weight was comparable across the different ovarian response groups (P = 0.127 and P = 0.19, respectively). Finally, the occurrence of adverse obstetric outcomes did not differ among the ovarian response categories. Multivariate regression analysis revealed that the number of oocytes was not associated with neonatal birth weight. CONCLUSIONS: No association was found between ovarian response and adverse perinatal outcomes in antagonist IVF and intracytoplasmic sperm injection cycles. Future, larger scale and prospectively designed investigations are needed to validate these results.

Impact of Serum Estradiol Levels Prior to Progesterone Administration in Artificially Prepared Frozen Embryo Transfer Cycles.

Background: The need for endocrine monitoring in artificial cycles for frozen embryo transfer (FET) remains unclear and, more specifically, the value of the late-proliferative phase serum estradiol (E2) levels is with conflicting evidence in current literature. Objective: To investigate whether artificial FET cycles require endocrine monitoring for the serum E2 level prior to initiation of exogenous progesterone administration after an endometrial thickness of 6.5 mm has been reached. Design: One thousand two hundred and twenty-two (n = 1,222) artificial FETs performed in a tertiary center between 2010 and 2015 were subdivided into 3 groups according to the following late-proliferative serum E2 level percentiles: ≤p10 (E2 ≤144 pg/ml; n = 124), p11-p90 (E2 from 145 to 438 pg/ml; n = 977) and >p90 (E2 >439 pg/ml; n = 121). A mixed-effects multilevel multivariable regression analysis was performed to assess the potential effect of the late-proliferative E2 level on the live birth rate (LBR). Results: The level of late-proliferative circulating E2 showed no significant difference in terms of LBR after FET. Specifically, the multivariable regression model demonstrated a LBR of 19.5% for the p11-p90 reference group, compared to 24.4% for the ≤p10 (p = 0.251) and 19.5% for the >p90 group (p = 0.989). Conclusion: In this large retrospective dataset, no association was observed between late-proliferative phase serum E2 levels and LBR following FET in artificially prepared cycles. Although, caution is warranted due to the retrospective nature of the analysis and the potential for unmeasured confounding, we argue that monitoring of the late-proliferative serum E2 levels and using them to guide clinical decision-making (e.g., medication step-up, cycle prolongation or cancelation) may be of questionable value.

Modified natural cycle IVF versus conventional stimulation in advanced-age Bologna poor responders.

RESEARCH QUESTION: Do ongoing pregnancy rates (OPR) differ between modified natural cycle IVF (MNC-IVF) and conventional high-dose ovarian stimulation (HDOS) in advanced-age Bologna poor responders? DESIGN: This was a retrospective cohort study including patients with poor ovarian response (POR) attending a tertiary referral university hospital from 1 January 2011 to 1 March 2017. All women who fulfilled the Bologna criteria for POR and aged ≥40 years who underwent their first intracytoplasmic sperm injection (ICSI) cycle in the study centre were included. RESULTS: In total, 476 advanced-age Bologna poor responder patients were included in the study: 189 in the MNC-IVF group and 287 in the HDOS group. OPR per patient were significantly lower in the MNC-IVF group (5/189, 2.6%) compared with the HDOS group (29/287, 10.1%) (P = 0.002). However, after adjustment for relevant confounders (number of oocytes and presence of at least one top-quality embryo), the multivariate logistic regression analysis showed that the type of treatment strategy (HDOS versus MNC-IVF) was not significantly associated with OPR (odds ratio 2.56, 95% confidence interval 0.9-7.6). CONCLUSIONS: In advanced-age Bologna poor responders, MNC-IVF, which is a more patient-friendly approach, could be a reasonable alternative in this difficult-to-treat group of women.

Added value today of hormonal measurements in ovarian stimulation in gonadotropin-releasing hormone antagonist treatment cycle.

PURPOSE OF REVIEW: Traditional approach of ovarian stimulation monitoring for in-vitro fertilization involves transvaginal sonography and serum estradiol measurements. Accumulating evidence has shown that hormonal evaluations during ovarian stimulation allow individual cycle optimization, moving away from only predicting the risk of ovarian hyperstimulation syndrome, but in addition assessing the impact of ovarian stimulation on endometrial receptivity, quality of oocytes, and subsequently embryos. The purpose of this review is to discuss the relevance and added value of hormonal monitoring during ovarian stimulation in gonadotropin-releasing hormone antagonist cycles where most of the advances have occurred. RECENT FINDINGS: Basal hormonal status, particularly estradiol, progesterone, and luteinizing hormone are instrumental in prediction of the patients with poor prognosis. Estradiol levels on the day of trigger are less sensitive in predicting ovarian hyperstimulation syndrome then the number of follicles more than 11 mm in diameter. Progesterone elevation on the day of trigger is associated with lower pregnancy rates. The gold standard treatment for progesterone elevation is to adopt a freeze-all strategy when the threshold of 1.50 ng/ml is exceeded. The effect of progesterone elevation on embryo quality remains to be confirmed by more trials. SUMMARY: Endocrine monitoring during ovarian stimulation allows fine-tuning of the physiology of the stimulated cycle and thereby increases the chances of successful treatment outcome.

Processing and selection of surgically-retrieved sperm for ICSI: a review.

Although the technique of intracytoplasmic sperm injection (ICSI) has been a revolution in the alleviation of male infertility, the use of testicular sperm for ICSI was a formerly unseen breakthrough in the treatment of the azoospermic man with primary testicular failure. At the clinical level, different procedures of testicular sperm retrieval (conventional TESE, micro-TESE, FNA/TESA, MESA, PESA) are being performed, the choice is mainly based on the cause of azoospermia (obstructive versus non-obstructive) and the surgeon's skills. At the level of the IVF laboratory, mechanical procedures to harvest the sperm from the tissue may be combined with enzymatic treatment in order to increase the sperm recovery rates. A number of techniques have been developed for viable sperm selection in males with only immotile testicular sperm available. However, large, well-designed studies on the benefit and safety of one over the other technique are lacking. Despite all the available methods and combinations of laboratory procedures which have a common goal to maximize sperm recovery from testicular samples, a large proportion of NOA patients fail to father a genetically own child. Advanced technology application may improve recovery rates by detection of the testicular foci with active spermatogenesis and/or identification of the rare individual sperm in the testicular suspensions. On the other hand, in vitro spermatogenesis or sperm production from embryonic stem cells or induced pluripotent stem cells might be future options. The present review summarizes the available strategies which aim to maximize sperm recovery from surgically retrieved samples.

Bien que la technique d'injection intra cytoplasmique d'un spermatozoïde (ICSI) ait constitué une révolution dans le soulagement de l'infertilité masculine, l'utilisation de spermatozoïdes testiculaires lors de l'ICSI fut une découverte capitale auparavant inaperçue du traitement de l'homme azoospermique par altération testiculaire primaire. Au plan clinique, différentes procédures de recueil des spermatozoïdes testiculaires sont réalisées (TESE conventionnelle, micro-TESE, FNA/TESA, MESA, PESA) dont le choix dépend principalement de la cause de l'azoospermie (obstructive versus non obstructive) et des compétences du chirurgien. Au plan du laboratoire de fécondation in vitro, des procédures mécaniques d'extraction des spermatozoïdes du tissu peuvent être combinées avec un traitement enzymatique dans l'objectif d'accroitre le taux de récupération de spermatozoïdes. Plusieurs techniques ont été développées pour sélectionner des spermatozoïdes vivants chez les hommes n'ayant que des spermatozoïdes testiculaires immobiles. Toutefois, il manque des études bien construites et à effectifs consistants portant sur le bénéfice et l'innocuité d'une technique par rapport aux autres. Malgré toutes les méthodes et combinaisons de procédures de laboratoire disponibles qui ont comme objectif commun d'accroitre au maximum la récupération de spermatozoïdes dans les échantillons testiculaires, un grand nombre de patients avec NOA échouent à obtenir un enfant qui soit génétiquement leur. L'utilisation d'une technologie de pointe devrait améliorer les taux de récupération par la détection de foyers testiculaires ayant une spermatogenèse active et/ou l'identification de rare spermatozoïde isolé dans les suspensions testiculaires. D'autre part, la spermatogenèse in vitro ou la production de spermatozoïdes à partir de cellules souches embryonnaires ou de cellules souches pluripotentes induites pourraient constituer de futures options. Le présent article de revue résume les stratégies disponibles qui visent à maximiser la récupération de spermatozoïdes dans des échantillons extraits chirurgicalement.

Bien que la technique d'injection intra cytoplasmique d'un spermatozoïde (ICSI) ait constitué une révolution dans le soulagement de l'infertilité masculine, l'utilisation de spermatozoïdes testiculaires lors de l'ICSI fut une découverte capitale auparavant inaperçue du traitement de l'homme azoospermique par altération testiculaire primaire. Au plan clinique, différentes procédures de recueil des spermatozoïdes testiculaires sont réalisées (TESE conventionnelle, micro-TESE, FNA/TESA, MESA, PESA) dont le choix dépend principalement de la cause de l'azoospermie (obstructive versus non obstructive) et des compétences du chirurgien. Au plan du laboratoire de fécondation in vitro, des procédures mécaniques d'extraction des spermatozoïdes du tissu peuvent être combinées avec un traitement enzymatique dans l'objectif d'accroitre le taux de récupération de spermatozoïdes. Plusieurs techniques ont été développées pour sélectionner des spermatozoïdes vivants chez les hommes n'ayant que des spermatozoïdes testiculaires immobiles. Toutefois, il manque des études bien construites et à effectifs consistants portant sur le bénéfice et l'innocuité d'une technique par rapport aux autres. Malgré toutes les méthodes et combinaisons de procédures de laboratoire disponibles qui ont comme objectif commun d'accroitre au maximum la récupération de spermatozoïdes dans les échantillons testiculaires, un grand nombre de patients avec NOA échouent à obtenir un enfant qui soit génétiquement leur. L'utilisation d'une technologie de pointe devrait améliorer les taux de récupération par la détection de foyers testiculaires ayant une spermatogenèse active et/ou l'identification de rare spermatozoïde isolé dans les suspensions testiculaires. D'autre part, la spermatogenèse in vitro ou la production de spermatozoïdes à partir de cellules souches embryonnaires ou de cellules souches pluripotentes induites pourraient constituer de futures options. Le présent article de revue résume les stratégies disponibles qui visent à maximiser la récupération de spermatozoïdes dans des échantillons extraits chirurgicalement.

Toxicity of twenty-two plant essential oils against pathogenic bacteria of vegetables and mushrooms.

ASBTRACT Toxicity of twenty-two essential oils to three bacterial pathogens in different horticultural systems: Xanthomonas campestris pv. phaseoli (causing blight of bean), Clavibacter michiganensis subsp. michiganensis (bacterial wilt and canker of tomato), and Pseudomonas tolaasii (causal agent of bacterial brown blotch on cultivated mushrooms) was tested. Control of bacterial diseases is very difficult due to antibiotic resistance and ineffectiveness of chemical products, to that essential oils offer a promising alternative. Minimal inhibitory and bactericidal concentrations are determined by applying a single drop of oil onto the inner side of each plate cover in macrodilution assays. Among all tested substances, the strongest and broadest activity was shown by the oils of wintergreen (Gaultheria procumbens), oregano (Origanum vulgare), and lemongrass (Cymbopogon flexuosus. Carvacrol (64.0-75.8%) was the dominant component of oregano oils, while geranial (40.7%) and neral (26.7%) were the major constituents of lemongrass oil. Xanthomonas campestris pv. phaseoli was the most sensitive to plant essential oils, being susceptible to 19 oils, while 11 oils were bactericidal to the pathogen. Sixteen oils inhibited the growth of Clavibacter michiganensis subsp. michiganensis and seven oils showed bactericidal effects to the pathogen. The least sensitive species was Pseudomonas tolaasii as five oils inhibited bacterial growth and two oils were bactericidal. Wintergreen, oregano, and lemongrass oils should be formulated as potential biochemical bactericides against different horticultural pathogens.

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.

Fruit Phenolic Composition of Different Elderberry Species and Hybrids.

UNLABELLED: The aim of this study was to investigate a detailed composition and content of phenolic compounds in fruits of 4 elderberry species (Sambucus nigra, S. cerulea, S. ebulus, and S racemosa) and 8 interspecific hybrids. Hydroxycinnamic acids (HCAs) represented the major share of phenolics in analyzed elderberries; caffeoylquinic and p-coumaroylquinic acids were most abundant. Flavanols (catechin, epicatechin, and different procyanidins) were the second major phenolic group detected in range from 2% to 30% of total analyzed phenolics. From the group of flavonols, 13 different quercetin glycosides, 7 kaempferol glycosides, and 8 isorhamnetin glycosides have been quantified. Rutin was the major flavonol in all studied genotypes. S. ebulus was characterized by the highest level of total HCAs, catechin, epicatechin, and most flavonols. Some elderberry hybrids, for example JA × RAC, CER × NI, and JA × (JA × NI), are perspective for further studies because they have high content of phenolic compounds. The results of research could contribute to breed cultivars, which may prove interesting for food-processing industries. PRACTICAL APPLICATION: Different levels of phenolic compounds have been measured in fruit of analyzed elderberry species and interspecific hybrids. Natural evaluation of elderberry genetic resources, the identification of phenolic compounds, and assessment of their properties are of great interest to breeders and directly valid for pharmaceutical and food industry.

Fungicide sensitivity of Trichoderma spp. from Agaricus bisporus farms in Serbia.

Trichoderma species, the causal agents of green mould disease, induce great losses in Agaricus bisporus farms. Fungicides are widely used to control mushroom diseases although green mould control is encumbered with difficulties. The aims of this study were, therefore, to research in vitro toxicity of several commercial fungicides to Trichoderma isolates originating from Serbian and Bosnia-Herzegovina farms, and to evaluate the effects of pH and light on their growth. The majority of isolates demonstrated optimal growth at pH 5.0, and the rest at pH 6.0. A few isolates also grew well at pH 7. The weakest mycelial growth was noted at pH 8.0-9.0. Generally, light had an inhibitory effect on the growth of tested isolates. The isolates showed the highest susceptibility to chlorothalonil and carbendazim (ED50 less than 1 mg L(-1)), and were less sensitive to iprodione (ED50 ranged 0.84-6.72 mg L(-1)), weakly resistant to thiophanate-methyl (ED50 = 3.75-24.13 mg L(-1)), and resistant to trifloxystrobin (ED50 = 10.25-178.23 mg L(-1)). Considering the toxicity of fungicides to A. bisporus, carbendazim showed the best selective toxicity (0.02), iprodione and chlorothalonil moderate (0.16), and thiophanate-methyl the lowest (1.24), while trifloxystrobin toxicity to A. bisporus was not tested because of its inefficiency against Trichoderma isolates.

Investigation of anthocyanin profile of four elderberry species and interspecific hybrids.

A total of 19 different anthocyanins have been detected in four elderberry species and eight hybrids and quantified with the use of HPLC-MS(n). The profile and content levels of anthocyanins varied considerably among the analyzed elderberry species and hybrids. Cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside were the most abundant anthocyanins in Sambucus nigra fruits. On the other hand, the prevalent anthocyanin in S. javanica hybrids was identified as cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside. The highest content of total analyzed anthocyanins (TAA) was determined in berries of the interspecific hybrid S. javanica × S. racemosa, followed by S. nigra, (S. javanica × S. nigra) × cv. Black Beauty, and (S. javanica × S. nigra) × S. cerulea. Berries of S. nigra var. viridis contained significantly lower levels of TAA. Our results provide novel information for nutritional research in addition to breeding programs, which strive to create new hybrids or cultivars with enhanced levels of bioactive components.

Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A.

The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell.

Severe ovarian hyperstimulation syndrome after gonadotropin-releasing hormone (GnRH) agonist trigger and "freeze-all" approach in GnRH antagonist protocol.

OBJECTIVE: To report two cases with GnRH agonist triggering and a freeze-all approach in a GnRH antagonist protocol resulting in the development of severe ovarian hyperstimulation syndrome (OHSS), requiring hospitalization and peritoneal drainage. DESIGN: Two case reports. SETTING: A tertiary referral center and an obstetrics and gynecology department of a hospital. PATIENT(S): Case 1 and case 2: severe OHSS with abdominal distension, ascites development, and hemoconcentration. INTERVENTION(S): Case 1 and case 2: diagnosed by clinical, hematologic, and ultrasound findings. Hospitalization, IV infusion, and peritoneal drainage. MAIN OUTCOME MEASURE(S): Symptomatic treatment and prevention of further complication. RESULT(S): Complete recovery. CONCLUSION(S): Two cases of severe OHSS after GnRH agonist trigger in a GnRH antagonist protocol without the administration of any hCG for luteal-phase support. Clinicians have to be aware that even the sequential approach to ovarian stimulation with a freeze-all attitude does not completely eliminate OHSS in all patients.

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function.

UNLABELLED: Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE: This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.

Does HPV type affect outcome in oropharyngeal cancer?

BACKGROUND: An epidemic of human papillomavirus (HPV)-related oropharyngeal squamous cell cancer (OPSCC) has been reported worldwide largely due to oral infection with HPV type-16, which is responsible for approximately 90% of HPV-positive cases. The purpose of this study was to determine the rate of HPV-positive oropharyngeal cancer in Southwestern Ontario, Canada. METHODS: A retrospective search identified ninety-five patients diagnosed with OPSCC. Pre-treatment biopsy specimens were tested for p16 expression using immunohistochemistry and for HPV-16, HPV-18 and other high-risk subtypes, including 31,33,35,39,45,51,52,56,58,59,67,68, by real-time qPCR. RESULTS: Fifty-nine tumours (62%) were positive for p16 expression and fifty (53%) were positive for known high-risk HPV types. Of the latter, 45 tumors (90%) were identified as HPV-16 positive, and five tumors (10%) were positive for other high-risk HPV types (HPV-18 (2), HPV-67 (2), HPV-33 (1)). HPV status by qPCR and p16 expression were extremely tightly correlated (p < 0.001, Fishers exact test). Patients with HPV-positive tumors had improved 3-year overall (OS) and disease-free survival (DFS) compared to patients with HPV-negative tumors (90% vs 65%, p = 0.001; and 85% vs 49%, p = 0.005; respectively). HPV-16 related OPSCC presented with cervical metastases more frequently than other high-risk HPV types (p = 0.005) and poorer disease-free survival was observed, although this was not statistically significant. CONCLUSION: HPV-16 infection is responsible for a significant proportion of OPSCC in Southwestern Ontario. Other high-risk subtypes are responsible for a smaller subset of OPSCC that present less frequently with cervical metastases and may have a different prognosis.

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor.

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle.

In vitro activity of antimicrobial agents against Pseudomonas tolaasii, pathogen of cultivated button mushroom.

In vitro antibacterial activity tests of seven biofungicides (Ekstrasol, Bisolbisan, Bisolbifit, Serenade, Sonata, Timorex, F-Stop) and two disinfectants (colloidal silver alone and in combination with hydrogen peroxide) against the Pseudomonas tolaasii strain (NS3B6) were carried out by the disc-diffusion, broth microdilution and broth macrodilution method. Biofungicides tested in this study did not exhibit any antimicrobial activity in neither one of the methods used. Disc diffusion method revealed high sensitivity of the tested P. tolaasii strain to Ecocute based on colloidal silver and hydrogen peroxide. Both microdilution and macrodilution methods identified the same MICs and MBCs of Ecocute (0.19 mg/L) for P. tolaasii strain. MICs and MBCs values of silver alone were much higher (10 mg/L) compared to silver in combination with hydrogen peroxide.