Essential Oils of Two Subspecies of Satureja montana L. against Gastrointestinal Parasite Anisakis simplex and Acetylcholinesterase Inhibition.

The increasing presence of Anisakis spp. in fish is having significant implications for public health due to a rise in cases of anisakiasis. Given this situation, there is a critical need to develop new strategies to fight this parasite. Satureja montana L., commonly known as savory, is a plant recognized in folk medicine for its therapeutic activity, such as being antispasmodic and digestive, among other properties. The aim of this study was to assess the nematicide activity against A. simplex larvae of the essential oil from two varieties of S. montana (subsp. montana (SMM) and variegata (SMV)). The essential oils were obtained via hydro-distillation of the flowering aerial parts. In vitro assays demonstrated the complete inactivation of anisakis larvae after 24 h when exposed to both essential oils, along with a significant reduction in their penetration capacity. Moreover, both essential oils showed an inhibitory effect on acetylcholinesterase (AChE). No differences between the subspecies were observed in any of the assays. Hence, the nematicidal activity of essential oils could be attributed to their capacity to inhibit AChE. These findings suggest the potential of S. montana essential oil for therapeutic and food industry applications.

Cytosolic delivery of peptidic STAT3 SH2 domain inhibitors.

The signal transducer and activator of transcription 3 (STAT3) protein is constitutively activated in several cancers. STAT3 activity can be blocked by inhibiting its Src Homology 2 (SH2) domain, but phosphotyrosine and its isosteres have poor bioavailability. In this work, we develop peptide-based inhibitors of STAT3-SH2 by combining chemical strategies that have proven effective for targeting other SH2 domains. These strategies include a STAT3-specific selectivity sequence, non-hydrolyzable phosphotyrosine isosteres, and a high-efficiency cell-penetrating peptide. Peptides that combined these three strategies had substantial biological stability and cytosolic delivery, as measured using highly quantitative cell-based assays. However, these peptides did not inhibit STAT3 activity in cells. By comparing in vitro binding affinity, cell penetration, and proteolytic stability, this work explores the delicate balance of factors that contribute to biological activity for peptidic inhibitors of STAT3.

Efficacy of Origanum syriacum Essential Oil against the Mosquito Vector Culex quinquefasciatus and the Gastrointestinal Parasite Anisakis simplex, with Insights on Acetylcholinesterase Inhibition.

Developing effective and eco-friendly antiparasitic drugs and insecticides is an issue of high importance nowadays. In this study, we evaluated the anthelminthic and insecticidal potential of the leaf essential oil obtained from Origanum syriacum against the L3 larvae of the parasitic nematode Anisakis simplex and larvae and adults of the mosquito Culex quinquefasciatus. Tests on A. simplex were performed by standard larvicidal and penetration assays, while mosquito toxicity was assessed relying on larvicidal, tarsal contact, and fumigation tests. To shed light on the possible mode of action, we analyzed the oil impact as acetylcholinesterase (AChE) inhibitor. This oil was particularly active on L3 larvae of A. simplex, showing a LC50 of 0.087 and 0.067 mg mL-1 after 24 and 48 h treatment, respectively. O. syriacum essential oil was highly effective on both larvae and adults of C. quinquefasciatus, showing LC50 values of 32.4 mg L-1 and 28.1 µg cm-2, respectively. Its main constituent, carvacrol, achieved larvicidal LC50(90) of 29.5 and 39.2 mg L-1, while contact toxicity assays on adults had an LC50(90) of 25.5 and 35.8 µg cm-2, respectively. In fumigation assays, the LC50 was 12.1 µL L-1 after 1 h and decreased to 1.3 µL L-1 in 24 h of exposure. Similarly, the fumigation LC50 of carvacrol was 8.2 µL L-1 after 1 h of exposure, strongly decreasing to 0.8 µL L-1 after 24 h of exposure. These results support the folk usage of Lebanese oregano as an antiparasitic agent, providing new insights about its utilization for developing new effective and eco-friendly nematocidal and insecticidal products.

Anti-HSV-2 activity of Terminalia chebula Retz extract and its constituents, chebulagic and chebulinic acids.

BACKGROUND: Development of new and effective therapeutics for sexually transmitted herpes simplex virus-2 (HSV-2) infection is important from public health perspective. With an aim to identify natural products from medicinal plants, in the present study, the potential of Terminalia chebula Retz was investigated for its activity against HSV-2. METHODS: Fruits of Terminalia chebula Retz were used to prepare 50% ethanolic extract. In addition, chebulagic acid and chebulinic acid both purified from T. chebula were also used. The extract as well as purified compounds were first used to determine their in vitro cytotoxicity on Vero cells by MTT assay. T. chebula extract, chebulagic acid, chebulinic acid along with acyclovir were subsequently assessed for direct anti-viral activity, and their ability to inhibit attachment and penetration of HSV-2 to the Vero cells. In addition, their anti-HSV-2 activity was also determined by in vitro post-infection plaque reduction assay. RESULTS: Cytotoxicity assay using Vero cells revealed CC50 = 409.71 ± 47.70 µg/ml for the extract whereas chebulagic acid and chebulinic acid showed more than 95% cell viability up to 200 µg/ml. The extract from T. chebula (IC50 = 0.01 ± 0.0002 µg/ml), chebulagic (IC50 = 1.41 ± 0.51 µg/ml) and chebulinic acids (IC50 = 0.06 ± 0.002 µg/ml) showed dose dependent potent in vitro direct anti-viral activity against HSV-2. These also effectively prevented the attachment as well as penetration of the HSV-2 to Vero cells. In comparison, acyclovir showed poor direct anti-viral activity and failed to significantly (p > 0.05) prevent the attachment as well as penetration of HSV-2 to Vero cells when tested upto 50 µg/ml. However, in post-infection plaque reduction assay, T. chebula extract, chebulagic and chebulinic acids showed IC50 values of 50.06 ± 6.12, 31.84 ± 2.64, and 8.69 ± 2.09 µg/ml, respectively, which were much lower than acyclovir (71.80 ± 19.95 ng/ml). CONCLUSIONS: The results presented herein suggest that T. chebula extract, chebulagic and chebulinic acids have higher direct antiviral activity against HSV-2 and efficacy to inhibit virus attachment and penetration to the host cells as compared to acyclovir. However, acyclovir is more potent to inhibit post-infection virus replication. Hence, T. chebula may be a useful candidate for developing alternative therapy for prevention of sexually transmitted HSV-2 infection. ᅟ.

Steinernema feltiae Intraspecific Variability: Infection Dynamics and Sex-Ratio.

Entomopathogenic nematodes (EPNs) from the Heterorhabditidae and Steinernematidae families are well-known biocontrol agents against numerous insect pests. The infective juveniles (IJs) are naturally occurring in the soil and their success in locating and penetrating the host will be affected by extrinsic/intrinsic factors that modulate their foraging behavior. Characterizing key traits in the infection dynamics of EPNs is critical for establishing differentiating species abilities to complete their life cycles and hence, their long-term persistence, in different habitats. We hypothesized that phenotypic variation in traits related to infection dynamics might occur in populations belonging to the same species. To assess these intraspecific differences, we evaluated the infection dynamics of 14 populations of Steinernema feltiae in two experiments measuring penetration and migration in sand column. Intraspecific variability was observed in the percentage larval mortality, time to kill the insect, penetration rate, and sex-ratio in both experiments (P < 0.01). Larval mortality and nematode penetration percentage were lower in migration experiments than in penetration ones in most of the cases. The sex-ratio was significantly biased toward female-development dominance (P < 0.05). When the populations were grouped by habitat of recovery (natural areas, crop edge, and agricultural groves), nematodes isolated in natural areas exhibited less larval mortality and penetration rates than those from some types of agricultural associated soils, suggesting a possible effect of the habitat on the phenotypic plasticity. This study reinforces the importance of considering intraspecific variability when general biological and ecological questions are addressed using EPNs.

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels.

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

In silico Docking Analysis for Blocking JUNO-IZUMO1 Interaction Identifies Two Small Molecules that Block in vitro Fertilization.

Combined hormone drugs are the basis for orally administered contraception. However, they are associated with severe side effects that are even more impactful for women in developing countries, where resources are limited. The risk of side effects may be reduced by non-hormonal small molecules which specifically target proteins involved in fertilization. In this study, we present a virtual docking experiment directed to discover molecules that target the crucial fertilization interactions of JUNO (oocyte) and IZUMO1 (sperm). We docked 913,000 molecules to two crystal structures of JUNO and ranked them on the basis of energy-related criteria. Of the 32 tested candidates, two molecules (i.e., Z786028994 and Z1290281203) demonstrated fertilization inhibitory effect in both an in vitro fertilization (IVF) assay in mice and an in vitro penetration of human sperm into hamster oocytes. Despite this clear effect on fertilization, these two molecules did not show JUNO-IZUMO1 interaction blocking activity as assessed by AVidity-based EXtracellular Interaction Screening (AVEXIS). Therefore, further research is required to determine the mechanism of action of these two fertilization inhibitors.

Throttling Growth Speed: Evaluation of aux1-7 Root Growth Profile by Combining D-Root system and Root Penetration Assay.

Directional root growth control is crucial for plant fitness. The degree of root growth deviation depends on several factors, whereby exogenous growth conditions have a profound impact. The perception of mechanical impedance by wild-type roots results in the modulation of root growth traits, and it is known that gravitropic stimulus influences distinct root movement patterns in concert with mechanoadaptation. Mutants with reduced shootward auxin transport are described as being numb towards mechanostimulus and gravistimulus, whereby different growth conditions on agar-supplemented medium have a profound effect on how much directional root growth and root movement patterns differ between wild types and mutants. To reduce the impact of unilateral mechanostimulus on roots grown along agar-supplemented medium, we compared the root movement of Col-0 and auxin resistant 1-7 in a root penetration assay to test how both lines adjust the growth patterns of evenly mechanostimulated roots. We combined the assay with the D-root system to reduce light-induced growth deviation. Moreover, the impact of sucrose supplementation in the growth medium was investigated because exogenous sugar enhances root growth deviation in the vertical direction. Overall, we observed a more regular growth pattern for Col-0 but evaluated a higher level of skewing of aux1-7 compared to the wild type than known from published data. Finally, the tracking of the growth rate of the gravistimulated roots revealed that Col-0 has a throttling elongation rate during the bending process, but aux1-7 does not.

Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative.

Enterovirus A71 (EV-A71) infection has emerged as a significant public health concern atthe global level. Epidemic events of EV-A71 have been reported worldwide, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat. In recent years, widespread A71 enterovirus also occurred in European countries. EV-A71 infection causes hand-foot-mouth disease (HFMD), herpangina, and fever. However, it can sometimes induce a variety of neurological complications, including encephalitis, aseptic meningitis, pulmonary edema, and acute flaccid paralysis. We identified new benzimidazole derivatives and described their in vitro cytotoxicity and broad-spectrum anti-enterovirus activity. Among them, derivative 2b resulted in interesting activity against EV-A71, and therefore it was selected for further investigations. Compound 2b proved to be able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC50 of 3 µM. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis when treated with 2b at 20 and 80 µM. Compound 2b reduced viral adsorption to Vero-76 cells, and when evaluated in a time-of-addition assay, the derivative had the highest effect when added during the infection period. Moreover, derivative 2b reduced viral penetration into host cells. Besides, 2b did not affect intestinal monolayers permeability, showing no toxic effects. A detailed insight into the efficacy of compound 2b against EV-A71 showed a dose-dependent reduction in the viral titer, also at low concentrations. Mechanism of action investigations suggested that our derivative can inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. Pharmacokinetic and toxicity predictions validated compound 2b as a good candidate for further in vivo assays.

The hamster egg penetration test may decrease intracytoplasmic sperm injection utilization while maintaining high conventional fertilization rates.

This was a cohort study of in vitro fertilization (IVF) subjects at the University of Utah, Salt Lake City (UT, USA) utilizing partner sperm. Cycles where both the hamster egg penetration test (HEPT) and semen analysis were performed within 2 years prior to IVF cycles were stratified into four groups based on a normal or an abnormal HEPT and morphology. The mean conventional and intracytoplasmic sperm injection (ICSI) fertilization rates were calculated in each group. We performed a univariate analysis on the primary outcome comparing clinically interesting subjects. We performed a cost-effectiveness analysis of a policy of HEPT versus universal ICSI in couples with an abnormal morphology. Among patients with a normal HEPT, there was no difference in the mean conventional fertilization rates between those with a normal and an abnormal morphology. There was no difference in the mean conventional fertilization rates between subjects with a normal morphology without a hamster test and those with a normal HEPT without a morphology assessment. In 1000 simulated cycles with an abnormal morphology, a policy of HEPT was cost saving compared to universal ICSI, yet produced similar fertilization rates. The HEPT is similar to the World Health Organization edition 5 (WHO-5) morphology in predicting successful conventional fertilization while allowing decreased utilization of ICSI. A policy of HEPT for males with abnormal morphology saves cost in selecting couples for a fertilization method.

Quantitative measurement of cytosolic penetration using the chloroalkane penetration assay.

A major barrier for drug development is ensuring molecules can access intracellular targets. This is especially true for biomolecules, which are notoriously difficult to deliver to the cytosol. Many current methods for measuring the internalization of therapeutic biomolecules are largely indirect and qualitative, and they do not offer information about subcellular localization. We recently reported a new assay, called the ChloroAlkane Penetration Assay (CAPA), that addresses some of the drawbacks of existing methods. CAPA is high-throughput, quantitative, and compartment-specific, and can be used to monitor cytosolic penetration over time and under a variety of culture conditions. We have used CAPA to investigate the cytosolic localization of peptides, proteins, and oligonucleotides. In this chapter, we discuss the materials, protocols, and troubleshooting necessary to perform CAPA and appropriately analyze the data. We end with a discussion about the applications and limitations of CAPA, and we speculate on the potential of the assay and its variations.

Improvement in pregnancy outcomes in couples with immunologically male infertility undergoing prednisolone treatment and conventional in vitro fertilization preceded by sperm penetration assay: a randomized controlled trial.

PURPOSE: Anti-sperm antibodies (ASA) in men impair not only sperm motility but also fertilization and conception. However, utilization of corticosteroids to suppress ASA has shown variable pregnancy outcomes. This controversy is also extended to include the usefulness of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in treatments of men with ASA. This study was therefore designed to define factors contributing to these inconsistent results. METHODS: Infertile men having ASA (n = 241) were randomly assigned for treatment with or without prednisolone for three cycles each of 21 days of their partner's menstrual cycles. Control and treated men underwent then human sperm penetration assay (SPA), of hamster oocytes, to diagnose men with impaired sperm fusogenic capacity. Men with positive or negative SPA results were admitted to conventional IVF or ICSI programs, respectively. RESULTS: Treated patients had improved sperm motility and progressive motility when compared to control patients (P < 0.001). Fertilization (P = 0.04), embryo cleavage (P = 0.01), and chemical (P = 0.02) and clinical (P = 0.04) pregnancy rates were higher in treated patients than in control patients undergoing conventional IVF but not ICSI cycles. CONCLUSIONS: Men with ASA may also have compromised sperm fusogenic capacity, which can mask the clinical significance of corticosteroids. Corticosteroid administration in men with ASA, but without compromised sperm fusogenic capacity, improves conventional IVF but not ICSI outcomes; the reason being that ICSI bypasses issues of compromised fusogenic capacity. Inclusion of SPA in infertility clinics that offer both conventional IVF and ICSI services may be useful to identify which patients with ASA benefit from corticosteroid treatments.

Cross-species fertilization: the hamster egg receptor, Juno, binds the human sperm ligand, Izumo1.

Fertilization is the culminating event in sexual reproduction and requires the recognition and fusion of the haploid sperm and egg to form a new diploid organism. Specificity in these recognition events is one reason why sperm and eggs from different species are not normally compatible. One notable exception is the unusual ability of zona-free eggs from the Syrian golden hamster (Mesocricetus auratus) to recognize and fuse with human sperm, a phenomenon that has been exploited to assess sperm quality in assisted fertility treatments. Following our recent finding that the interaction between the sperm and egg recognition receptors Izumo1 and Juno is essential for fertilization, we now demonstrate concordance between the ability of Izumo1 and Juno from different species to interact, and the ability of their isolated gametes to cross-fertilize each other in vitro. In particular, we show that Juno from the golden hamster can directly interact with human Izumo1. These data suggest that the interaction between Izumo1 and Juno plays an important role in cross-species gamete recognition, and may inform the development of improved prognostic tests that do not require the use of animals to guide the most appropriate fertility treatment for infertile couples.

Reproductive performance, semen quality, and fatty acid profile of spermatozoa in senescent broiler breeder roosters as enhanced by the long-term feeding of dried apple pomace.

Avian spermatozoa are characteristically high in polyunsaturated fatty acids which predispose them to lipoperoxidation, thereby diminishing their fertility. As a by-product, well-known for antioxidative properties, dried apple pomace (AP) was fed to aging Ross 308 breeder roosters (n=80) at the dietary inclusion levels of 0 (AP0), 10 (AP10), 20 (AP20), or 25 (AP25) percent for 14 consecutive weeks. Seminal traits were studied every other week. At the end of the trial, sperm fatty acids profile, seminal plasma total antioxidant capacity (TAC), fertility, hatchability, and hatchling quality were evaluated, using 240 artificially-inseminated young hens. Dietary AP increased sperm motility and livability and decreased the seminal content of thiobarbituric acid reactive species. Dietary treatment and age interacted to positively affect sperm concentration and sperm membrane integrity. Feeding AP was associated with an increase in C20:4(n-6), C22:4(n-6), and total unsaturated fatty acids percentages. Birds in the AP25 and AP20 treatment groups respectively showed 6 and 7% increases in fertility rate. Interestingly, a higher hatchability rate was found for AP20 group, associated with a higher number of sperm penetration holes in the perivitelline membrane and a lower rate of early embryonic mortality. However, hatchling quality was not affected by dietary AP. Overall, these data suggest that AP could remarkably improve several sperm characteristics, seminal TAC, fertility, and hatchability rate in aging breeder roosters. These improvements were also associated with a higher content of total unsaturated FA in the sperm plasma membrane. Future studies are needed to disclose the causal mechanisms involved.

Sperm functional tests.

Several semen parameters are used to discriminate the fertile male from the subfertile male. The most widely used parameters are sperm concentration, motility, progressive motility, and sperm morphology. Semen analysis is usually applied as described in the World Health Organization manual for semen analysis. In addition to a routine semen analysis, sperm functional tests have been described for many years, which in most cases are regarded as research tools and not part of the routine semen testing in an infertility clinic. In this review we report on the value of four sperm function tests: the sperm penetration assay, the sperm-zona pellucida binding tests, the acrosome reaction, and the hyaluronan binding assay. For each test we describe the current value, the indication for performing the test, how to interpret the results, and its therapeutic implications. Our data show that sperm functional assays are highly predictive of IVF outcome results and have the potential to assist in clinical decision making, especially to avoid the current long-standing treatment with IUI and to direct the patients to intracytoplasmic sperm injection without delay when sperm functional testing fails. We believe that advances in molecular biology techniques will allow us to develop simpler sperm function assays in the near future. This will undoubtedly help clinicians in optimizing male factor infertility diagnosis and treatment.

Daphne Genkwa sieb. Et zucc. Water-soluble extracts act on enterovirus 71 by inhibiting viral entry.

Dried flowers of Daphne genkwa Sieb. et Zucc. (Thymelaeaceae) are a Chinese herbal medicine used as an abortifacient with purgative, diuretic and anti-inflammatory activities. However, the activity of this medicine against enteroviral infections has not been investigated. The water-extract of dried buds of D. genkwa Sieb. et Zucc. (DGFW) was examined against various strains of enterovirus 71 (EV71) by neutralization assay, and its initial mode of action was characterized by time-of-addition assay followed by attachment and penetration assays. Pretreatment of DGFW with virus abolished viral replication, indicating that DGFW inhibits EV71 by targeting the virus. GFW exerts its anti-EV71 effects by inhibiting viral entry without producing cytotoxic side effects and thus provides a potential agent for antiviral chemotherapeutics.

Semen analysis and sperm function tests: How much to test?

Semen analysis as an integral part of infertility investigations is taken as a surrogate measure for male fecundity in clinical andrology, male fertility, and pregnancy risk assessments. Clearly, laboratory seminology is still very much in its infancy. In as much as the creation of a conventional semen profile will always represent the foundations of male fertility evaluation, the 5th edition of the World Health Organization (WHO) manual is a definitive statement on how such assessments should be carried out and how the quality should be controlled. A major advance in this new edition of the WHO manual, resolving the most salient critique of previous editions, is the development of the first well-defined reference ranges for semen analysis based on the analysis of over 1900 recent fathers. The methodology used in the assessment of the usual variables in semen analysis is described, as are many of the less common, but very valuable, sperm function tests. Sperm function testing is used to determine if the sperm have the biologic capacity to perform the tasks necessary to reach and fertilize ova and ultimately result in live births. A variety of tests are available to evaluate different aspects of these functions. To accurately use these functional assays, the clinician must understand what the tests measure, what the indications are for the assays, and how to interpret the results to direct further testing or patient management.

Hyaluronan binding assay (HBA) vs. sperm penetration assay (SPA): Can HBA replace the SPA test in male partner screening before in vitro fertilization?

OBJECTIVE: To determine if a less expensive, easier, and faster to perform HBA test is clinically equal to the more complicated, technically challenging and expensive SPA test as a reliable indicator of sperm fertilizing capacity. DESIGN: Prospective study. SETTING: Andrology laboratory within In Vitro Fertilization Program. PATIENT(S): Semen samples from 26 infertility couples were analyzed. Both, normal and male factor patients were included. INTERVENTION(S): Male partner screening with the HBA and the SPA tests. MAIN OUTCOME MEASURE(S): Relationship between HBA and SPA test results. RESULT(S): The data obtained in this study showed no statistically significant relationship between the HBA and SPA results. The mean HBA scores 76.3%, 61.3% and 76.8% were statistically not significantly different as compared to patients with negative (<5), grey zone (5-8) and for positive (>8) sperm capacitation index values. CONCLUSION(S): The HBA is not predictive of the results of the SPA. Therefore, HBA test does not reduce the need for and cannot replace the SPA test in male partner screening prior to infertility treatment.