In silico and In vitro Assessment of Dimeric Flavonoids (Brachydins) on Rhipicephalus microplus Glutathione S-transferase.

INTRODUCTION: Rhipicephalus microplus, an important cattle ectoparasite, is responsible for a substantial negative impact on the economy due to productivity loss. The emergence of resistance to widely used commercial acaricides has sparked efforts to explore alternative products for tick control. METHOD: To address this challenge, innovative solutions targeting essential tick enzymes, like glutathione S-transferase (GST), have gained attention. Dimeric flavonoids, particularly brachydins (BRAs), have demonstrated various biological activities, including antiparasitic effects. The objectives of this study were to isolate four dimeric flavonoids from Fridericia platyphylla roots and to evaluate their potential as inhibitors of R. microplus GST. RESULTS: In vitro assays confirmed the inhibition of R. microplus GST by BRA-G, BRA-I, BRA-J, and BRA-K with IC50 values of 0.075, 0.079, 0.075, and 0.058 mg/mL, respectively, with minimal hemolytic effects. Molecular docking of BRA-G, BRA-I, BRA-J, and BRA-K in a threedimensional model of R. microplus GST revealed predicted interactions with MolDock Scores of - 142.537, -126.831, -108.571, and -123.041, respectively. Both in silico and in vitro analyses show that brachydins are potential inhibitors of R. microplus GST. CONCLUSION: The findings of this study deepen our understanding of GST inhibition in ticks, affirming its viability as a drug target. This knowledge contributes to the advancement of treatment modalities and strategies for improved tick control.

An alkaloidal fraction of Annona tomentosa possesses anthelmintic activity and induces surface modifications in Haemonchus contortus.

The nematode Haemonchus contortus is, as a parasite, responsible for most mortality of small ruminants, causing significant economic losses. Numerous plant-derived compounds have exhibited promising anthelmintic activities against this nematode. Notably, the Annona genus stands out for demonstrated anthelmintic effects by extracts from several of its species against different nematodes. This study aimed to assess the effect of an Annona tomentosa fraction, rich in alkaloids, on H. contortus. This fraction, named Alk.F, is derived from the methanolic extract of the plant's stem bark. Chemical characterization of Alk.F was performed by liquid chromatography coupled with mass spectrometry. Among the nine predominant peaks obtained, seven alkaloids were identified: reticuline, reticuline N-oxide, reticuline N-oxide isomer, cyclanoline, asimilobine, tetrahydropalmatine and anonaine. Alk.F inhibited the larval development of H. contortus with an IC50 of 0.026 mg/mL, inhibited larval exsheathment with an IC50 of 0.38 mg/mL, and displayed low hemolytic activity towards sheep erythrocytes. Furthermore, atomic force microscopy revealed that Alk.F altered adhesive forces and the height profile on the surface of H. contortus larvae. In conclusion, A. tomentosa alkaloids alter the cuticle structure of H. contortus, inhibiting larval development and exsheathment, thus offering possibilities for contributing to the development of new anthelmintic drugs.

Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.

Introduction: Head and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches. Methods: In the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. Results: In vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor. Discussion: Hence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.

Actinic keratosis - lesions behind the mask.

INTRODUCTION: Actinic keratoses (AKs) are common skin lesions that arise in skin areas chronically exposed to ultraviolet (UV) radiation. They may progress to squamous cell carcinomas in 16% of cases within 1 year. Clinically, they present as erythematous scaly plaques and mainly affect face, neck, chest, back of the hands, shoulders and scalp. Cumulative exposure to UV radiation is the main risk factor. Other factors are advanced age, outdoor activities, geographic characteristics, exposure to artificial UV radiation and chronic skin inflammation. Many of these factors are often present in rural populations where agriculture remains important. METHODS/RESULTS: This presentation present the case of a 67-year-old male patient, who went to his Family Doctor for odynophagia with 2 days of evolution. He had hypertrophied and erythematous tonsils with purulent exudate and was medicated with amoxicillin-clavulanic acid 875+125 mg for 8 days with improvement of symptoms. To perform the observation of the oropharynx, he was asked to remove his face mask, which revealed an erythematous scaly lesion in the left malar region, suggestive of actinic keratosis. He was referred to Dermatology where cryotherapy of the lesion was performed with a favourable evolution without relapses. DISCUSSION: AKs are pre-malignant lesions. Rural populations are particularly at risk for their development. It is therefore essential to raise awareness for the use of protective measures as well as to investigate lesions already established. This case seeks to alert for the fact that the use of masks due to COVID-19 pandemic can hide pre-malignant lesions of the face with a consequent delay in diagnosis and treatment.

Anonaine from Annona crassiflora inhibits glutathione S-transferase and improves cypermethrin activity on Rhipicephalus (Boophilus) microplus (Canestrini, 1887).

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) is one of the most important ectoparasites of cattle, causing severe economic losses in tropical and subtropical regions of the world. The selection of resistance to the most commonly used commercial acaricides has stimulated the search for new products for tick control. The identification and development of drugs that inhibit key tick enzymes, such as glutathione S-transferase (GST), is a rational approach that has already been applied to other parasites than ticks. In this context, alkaloids such as anonaine display several biological activities, including an acaricidal effect. This study aimed to assess the specific inhibition of the R. microplus GST by anonaine, and analyze the effect on ticks when anonaine is combined with cypermethrin. For this purpose, a molecular docking analysis was performed using an R. microplus GST three-dimensional structure model with anonaine and compared with a human GST-anonaine complex. The absorption, distribution, metabolism, excretion, and toxicity properties of anonaine were also predicted. Then, for in vitro analyses, anonaine was isolated from Annona crassiflora (Martius, 1841) leaves. The inhibition of purified recombinant R. microplus GST (rRmGST) by anonaine and the effect of this alkaloid on cypermethrin efficacy towards R. microplus were assessed. Anonaine has a higher affinity to the tick enzyme than to the human enzyme in silico and has moderate toxicity, being able to inhibit, in vitro, rRmGST up to 37.5% in a dose-dependent manner. Although anonaine alone has no activity against R. microplus, it increased the cypermethrin effect on larvae, reducing the LC50 from 44 to 22 µg/mL. In conclusion, anonaine is a natural compound that can increase the effect of cypermethrin against R. microplus.

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression.

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h-1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h-1 and 2-fold higher for 128 L h-1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h-1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

Effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of Rhipicephalus microplus (Acari: Ixodidae).

The present study was conducted to evaluate the effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of larvae from two populations of R. microplus: Jaguar (tick population resistant to six classes of acaricides) and Porto Alegre (susceptible tick population). Carvacrol and thymol were tested at concentrations ranging from 0.14 to 5.0 mg mL-1 in both populations to determine the LC50. In addition, the LC1, LC25, and LC75 were estimated using the LC50 and HillSlope of each compound. Larvae of both populations of R. microplus were then treated with the LC1, LC25, LC50, and LC75 of each monoterpene, and those that survived were processed to evaluate the effects of the compounds on the antioxidant and detoxifying systems of larvae; these effects were assessed by determining the activity of the enzymes, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Larvae from the Jaguar population treated with different lethal concentrations of carvacrol and thymol displayed a dose-dependent increase in CAT, GPX, SOD, and GST after treatment with LC25. Further, larvae treated with the LC75 had the highest levels of enzyme activity for carvacrol (1.76 mg mL-1) and thymol (1.32 mg mL-1). CAT, GPX, SOD, and GST activity in Porto Alegre population larvae treated with carvacrol and thymol also increased significantly up to the LC50 of each monoterpene. However, at the LC75 of carvacrol and thymol, a decrease in the activity of all enzymes was observed for this tick population. These findings indicate that carvacrol and thymol induced increased activity of all evaluated enzymes at different lethal concentrations in R. microplus larvae from two populations. Such findings unveil the possible mechanisms of action of these natural acaricides.

A Large Cohort Study of SARS-CoV-2 Detection in Saliva: A Non-Invasive Alternative Diagnostic Test for Patients with Bleeding Disorders.

Diagnosis of SARS-CoV-2 infections is mostly based on the nasopharyngeal swabs (NPS). However, this collection is invasive and uncomfortable, especially for children and patients with coagulopathies, whose NPS collection often causes bleeding. Thus, the aim of this study was to evaluate the usefulness and accuracy of saliva for the diagnosis of COVID-19 in patients presenting bleeding disorders. Samples of NPS, oropharyngeal swabs (OPS), and saliva were collected simultaneously from 1159 hospitalized patients with hematological diseases and from 524 healthcare workers, both symptomatic and asymptomatic for SARS-CoV-2. All samples were evaluated for SARS-CoV-2 by qRT-PCR. SARS-CoV-2 was detected in NPS, OPS and saliva from 16.9%, 14.4% and 15.6% individuals, respectively. Tests in saliva showed sensitivity, specificity, and overall agreement of 73.3%, 96.9% and 92.7% (=0.74), respectively. Salivary tests had good accuracy (AUC = 0.7) for discriminating negative and positive qRT-PCR for SARS-CoV-2. Higher sensitivity was observed in symptomatic than in non-symptomatic patients, as well as in healthy subjects than in patients with hematological disease, in both OPS and saliva. The mean viral load in NPS was significantly higher than in OPS and in saliva samples (p < 0.001). Saliva is a good diagnostic tool to detect SARS-CoV-2, especially among patients symptomatic for COVID-19, and is a valuable specimen for mass screening of hospitalized patients with hematological diseases, especially for those that with bleeding disorders.

Comparison of Speech Recognition in Cochlear Implant Users with Different Speech Processors.

BACKGROUND: Speech recognition in noisy environments is a challenge for both cochlear implant (CI) users and device manufacturers. CI manufacturers have been investing in technological innovations for processors and researching strategies to improve signal processing and signal design for better aesthetic acceptance and everyday use. PURPOSE: This study aimed to compare speech recognition in CI users using off-the-ear (OTE) and behind-the-ear (BTE) processors. DESIGN: A cross-sectional study was conducted with 51 CI recipients, all users of the BTE Nucleus 5 (CP810) sound processor. Speech perception performances were compared in quiet and noisy conditions using the BTE sound processor Nucleus 5 (N5) and OTE sound processor Kanso. Each participant was tested with the Brazilian-Portuguese version of the hearing in noise test using each sound processor in a randomized order. Three test conditions were analyzed with both sound processors: (i) speech level fixed at 65 decibel sound pressure level in a quiet, (ii) speech and noise at fixed levels, and (iii) adaptive speech levels with a fixed noise level. To determine the relative performance of OTE with respect to BTE, paired comparison analyses were performed. RESULTS: The paired t-tests showed no significant difference between the N5 and Kanso in quiet conditions. In all noise conditions, the performance of the OTE (Kanso) sound processor was superior to that of the BTE (N5), regardless of the order in which they were used. With the speech and noise at fixed levels, a significant mean 8.1 percentage point difference was seen between Kanso (78.10%) and N5 (70.7%) in the sentence scores. CONCLUSION: CI users had a lower signal-to-noise ratio and a higher percentage of sentence recognition with the OTE processor than with the BTE processor.

Interactome analysis reveals endocytosis and membrane recycling of EpCAM during differentiation of embryonic stem cells and carcinoma cells.

Transmembrane epithelial cell adhesion molecule (EpCAM) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESCs). EpCAM displays spatiotemporal patterning during embryogenesis, tissue morphogenesis, cell differentiation, and epithelial-to-mesenchymal transition (EMT) in carcinomas. Potential interactors of EpCAM were identified in murine F9 teratoma cells using a stable isotope labeling with amino acids in cell culture-based proteomic approach (n = 77, enrichment factor >3, p value ≤ 0.05). Kyoto Encyclopedia of Genes and Genomes and gene ontology terms revealed interactions with regulators of endosomal trafficking and membrane recycling, which were further validated for Rab5, Rab7, and Rab11. Endocytosis and membrane recycling of EpCAM were confirmed in mF9 cells, E14TG2α ESC, and Kyse30 carcinoma cells. Reduction of EpCAM during mesodermal differentiation and TGFß-induced EMT correlated with enhanced endocytosis and block or reduction of recycling in ESCs and esophageal carcinoma cells. Hence, endocytosis and membrane recycling are means of regulation of EpCAM protein levels during differentiation of ESC and EMT induction in carcinoma cells.

Molluscicidal activity of monoterpenes and their effects on inhibition of acetylcholinesterase activity on Biomphalaria glabrata, an intermediate host of Schistosoma mansoni.

The molluscicidal action of essential oils have been attributed to the most prevalent terpene compounds. However, molluscicidal properties, mode of action, and toxicity to non-target organisms remain unclear. In this study, the molluscicidal potential of four monoterpenes (camphor, thymol, α-pinene, and 1,8-cineole) against the snail Biomphalaria glabrata, an intermediate host of Schistosoma mansoni, was analyzed. The molluscicide activity of each monoterpene was assessed by the standardized test of the World Health Organization (WHO) and the monoterpenes considered active against B. glabrata were analyzed as inhibitors of the enzymatic activity of acetylcholinesterase (AChE) extracted from snails. In addition, acute toxicity to non-target organisms was assessed against Danio rerio fish. The results show that camphor and 1,8-cineole monoterpenes did not induce snail mortality. Thymol and α-pinene were active against B. glabrata, inducing mortality in concentration-dependent patterns and showing a lethal effect in concentrations compatible with that recommended by the WHO (LC90 of 7.11 and LC90 10.34 µg ∙ mL-1, respectively). The toxic action of thymol and α-pinene on snails indicates that these monoterpenes may account for or largely contribute to the molluscicidal activity of essential oils that contain them as major compounds. Thymol and α-pinene inhibit the AChE of B. glabrata at concentrations higher than those used in the molluscicide test. These monoterpenes show low toxicity to non-target organisms compared to the commercial molluscicide niclosamide. Knowledge about monoterpene toxicity against B. glabrata contributes to its potential use in molluscicidal formulations and in alternatives to the control of snails that host intermediate S. mansoni, a crucial action in the prevention and transmission of schistosomiasis, a neglected tropical disease.

New Insights into Anthelmintic Mechanisms of Action of a Synthetic Peptide: An Ultrastructural and Nanomechanical Approach.

Resistant nematodes are not affected by the most common drugs commercially available. In the search for new anthelmintics, peptides have been investigated. Here, a linear synthetic peptide named RcAlb-PepIII bioinspired from the antimicrobial protein Rc-2S-Alb was designed, synthesized, and tested against barber pole worm Haemonchus contortus. The physicochemical properties of the peptide, the 3D structure model, the egg hatch inhibition, and larval development inhibition of H. contortus were carried out. Additionally, the ultrastructure of the nematode after treatment with the peptide was evaluated by atomic force microscopy. The RcAlb-PepIII inhibited the larval development of H. contortus with an EC50 of 90 µM and did not affect egg hatch. Atomic force microscopy reveals the high affinity of RcAlb-PepIII with the cuticle of H. contortus in the L2 stage. It also shows the deposition of RcAlb-PepIII onto the surface of the cuticle, forming a structure similar to a film that reduces the roughness and mean square roughness (Rq) of it. In conclusion, the bioinspired RcAlb-PepIII has the potential to be used as a new anthelmintic compound to control gastrointestinal nematode parasites.

Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus.

This study reports the action of essential oils (EO) from five plants on the activity of native and recombinant acetylcholinesterases (AChE) from Rhipicephalus microplus. Enzyme activity of native susceptible AChE extract (S.AChE), native resistant AChE extract (R.AChE), and recombinant enzyme (rBmAChE1) was determined. An acetylcholinesterase inhibition test was used to verify the effect of the EO on enzyme activity. EO from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var.dulcis inhibited the activity of S.AChE and R.AChE. Oils from the two Citrus species inhibited S.AChE and R.AChE in a similar way while showing greater inhibition on R.AChE. The oil from E. globulus inhibited native AChE, but no difference was observed between the S.AChE and R.AChE; however, 71% inhibition for the rBmAChE1 was recorded. Mentha piperita oil also inhibited S.AChE and R.AChE, but there was significant inhibition at the highest concentration tested. Cymbopogon winterianus oil did not inhibit AChE. Further studies are warranted with the oils from the two Citrus species that inhibited R.AChE because of the problem with R. microplus resistant to organophosphates, which target AChE. C. winterianus oil can be used against R. microplus populations that are resistant to organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.

High-resolution spatiotemporal pHe and pO2 imaging in head and neck and oesophageal carcinoma cells.

BACKGROUND: pO2 and pH are physiological parameters relevant for different processes in health and disease, including wound healing and cancer progression. Head and neck squamous cell carcinomas (HNSCC) and oesophageal squamous cell carcinomas (ESCC) have a high rate of local recurrence that is partly related to treatment-resistant residual tumour cells. Hence, novel diagnostic tools are required to visualise potential residual tumour cells and thereby improve treatment outcome for HNSCC and ESCC patients. We developed a device to spatiotemporally measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) to distinguish HNSCC and ESCC cells from healthy cells in vitro, exploiting general metabolic differences between cancer cells and healthy cells. METHODS: OCR and ECAR were measured via a newly developed device named STO2p-Q (SpatioTemporal O2 and pH Quantification) using the VisiSens technology based on ratiometric fluorescence imaging, facilitating spatiotemporal resolution. Results were confirmed using extracellular flux analyses (Seahorse technology). RESULTS: STO2p-Q is described and used to measure OCR and ECAR in HNSCC and ESCC cell lines and normal fibroblast and epithelial cells as components of the tumour microenvironment. OCR measurements showed differences amongst HNSCC and ESCC cell lines and between HNSCC/ESCC and normal cells, which on average had lower OCR than HNSCC/ESCC cells. Both OCR and ECAR measurements were independently verified using the Seahorse technology. Additionally, using STO2p-Q, HNSCC/ESCC, and normal cells could be spatially resolved with a resolution in the low millimetre range. CONCLUSIONS: We developed a method to spatiotemporally measure OCR and ECAR of cells, which has many potential in vitro applications and lays the foundation for the development of novel diagnostic tools for the detection of cancerous tissue in HNSCC and ESCC patients in vivo.

Protein-Ligand Docking Simulations with AutoDock4 Focused on the Main Protease of SARS-CoV-2.

BACKGROUND: The main protease of SARS-CoV-2 (Mpro) is one of the targets identified in SARS-CoV-2, the causative agent of COVID-19. The application of X-ray diffraction crystallography made available the three-dimensional structure of this protein target in complex with ligands, which paved the way for docking studies. OBJECTIVE: Our goal here is to review recent efforts in the application of docking simulations to identify inhibitors of the Mpro using the program AutoDock4. METHODS: We searched PubMed to identify studies that applied AutoDock4 for docking against this protein target. We used the structures available for Mpro to analyze intermolecular interactions and reviewed the methods used to search for inhibitors. RESULTS: The application of docking against the structures available for the Mpro found ligands with an estimated inhibition in the nanomolar range. Such computational approaches focused on the crystal structures revealed potential inhibitors of Mpro that might exhibit pharmacological activity against SARS-CoV-2. Nevertheless, most of these studies lack the proper validation of the docking protocol. Also, they all ignored the potential use of machine learning to predict affinity. CONCLUSION: The combination of structural data with computational approaches opened the possibility to accelerate the search for drugs to treat COVID-19. Several studies used AutoDock4 to search for inhibitors of Mpro. Most of them did not employ a validated docking protocol, which lends support to critics of their computational methodology. Furthermore, one of these studies reported the binding of chloroquine and hydroxychloroquine to Mpro. This study ignores the scientific evidence against the use of these antimalarial drugs to treat COVID-19.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Digital scoring of EpCAM and slug expression as prognostic markers in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) have poor clinical outcome owing to therapy resistance and frequent recurrences that are among others attributable to tumor cells in partial epithelial-to-mesenchymal transition (pEMT). We compared side-by-side software-based and visual quantification of immunohistochemistry (IHC) staining of epithelial marker EpCAM and EMT regulator Slug in n = 102 primary HNSCC to assess optimal analysis protocols. IHC scores incorporated expression levels and percentages of positive cells. Digital and visual evaluation of membrane-associated EpCAM yielded correlating scorings, whereas visual evaluation of nuclear Slug resulted in significantly higher overall scores. Multivariable Cox proportional hazard analysis defined the median EpCAM expression levels resulting from visual quantification as an independent prognostic factor of overall survival. Slug expression levels resulting from digital quantification were an independent prognostic factor of recurrence-free survival, locoregional recurrence-free survival, and disease-specific survival. Hence, we propose to use visual assessment for the membrane-associated EpCAM protein, whereas nuclear protein Slug assessment was more accurate following digital measurement.