Motility Assay Research Articles

Targeted dual-receptor phage cocktail against Cronobacter sakazakii: insights into phage-host interactions and resistance mechanisms

IntroductionCronobacter sakazakii is a notorious foodborne pathogen, frequently contaminating powdered infant formula and causing life-threatening diseases in infants. The escalating emergence of antibiotics-resistant mutants has led to increased interest in using bacteriophage as an alternative antimicrobial agent.MethodsTwo phages, CR8 and S13, were isolated from feces and soil samples and their morphology, physiology, and genomics were characterized. Phage receptor was determined using deletion mutants lacking flgK, rfaC, fhuA, btuB, lamb, or ompC genes, followed by complementation. Phage-resistant mutants were analyzed for phenotypic changes and fitness trade-offs using motility assays and Caco-2 cell invasion models.ResultsCR8 and S13 were identified as members of Caudoviricetes. Phage CR8 and phage S13 utilize flagella and LPS, respectively, to adhere to host cells. Bacterial challenge assay demonstrated delayed emergence of the resistant mutant as well as stronger lytic activity of a phage cocktail consisting of CR8 and S13 than the single phage treatment. Phenotypic analysis of the phage cocktail resistant strain, designated as CSR strain, revealed that the resistance resulted from the impaired receptor proteins for phage, such as defects in motility and alteration in LPS structure. CSR strain exhibited significant attenuation in invading human intestinal epithelial Caco-2 cells compared to WT cells.ConclusionThis study demonstrates that the development of the phage cocktail targeting distinct host receptors can serve as a promising antimicrobial strategy to effectively control C. sakazakii.

Blocking CXCR3B Expression Increases Tumor Aggressiveness in Hepatocellular Carcinoma.

CXCR3B has been positively involved in the inhibition of cancer and angiogenesis. The present study investigated the role of CXCR3B in a cell model of hepatocellular carcinoma, SK-Hep1. The blockade of CXCR3B expression in SK-Hep1 was investigated in terms of cell viability, cell cycle, and cell apoptosis using MTT assay and flow cytometry. In addition, the effect of blocking CXCR3B expression on cell migration and invasion was examined using scratch motility, transwell migration, and invasion assays. Furthermore, the cytotoxic effect of NK-92 cells against CXCR3B blocked SK-Hep1 was analyzed using the CytoTox96 assay, and the expression of NKp30+, NKG2D+, and NKG2C+ on NK-92 cells in a co-culture with SK-Hep1 was measured using flow cytometry. Blocking CXCR3B expression had no effect on the viability, cell cycle or apoptosis of SK-Hep1 cells. However, blockade of CXCR3B expression significantly increased the migratory and invasive ability of SK-Hep1 along with increased protein expression of slug, vimentin, and N-cadherin. CXCR3B blockade reduced the cytotoxicity of NK-92 against SK-Hep1 and inhibited the expression of activating receptors, NKp30+, NKG2D+, and NKG2C+ in NK-92 cells. CXCR3B may play a positive role in suppressing HCC by attenuating natural killer cell cytotoxicity against HCC.

Human Toll-like receptor activation by pathogenic Arcobacter species

The increase of Arcobacter spp. infection cases in humans, coupled with varying symptomatology, highlights the need to study the virulence mechanisms of these bacteria. Arcobacter butzleri can induce the release of several proinflammatory cytokines in human monocytic-derived macrophages, but the mechanism used to achieve this is still unclear. Therefore, we aimed to investigate the human innate immune response triggered by pathogenic Arcobacter spp., by studying the activation of the human Toll-like receptors (TLRs). Arcobacter skirrowii was the only species that showed the ability to activate all tested TLRs. Arcobacter cryaerophilus demonstrated to be able to activate TLR1/2, TLR4, and TLR2/6. A. butzleri hardly activated the TLRs, only TLR2/6 and TLR1/2 to a small extent. While all the Arcobacter species tested possess flagellum, as shown by motility assays and electron microscopy, only the flagellum of A. skirrowii was able to activate TLR5. The alignment of the flagellin amino acid data revealed that A. skirrowii shares a greater number of crucial amino acids for TLR5 recognition with the FliC of Salmonella than the other Arcobacter species, which might explain why A. skirrowii activates TLR5. Our results demonstrated that the activation of the different TLRs is Arcobacter species dependent, and there might be a correlation between the activation of the TLRs and the pathogenicity of the Arcobacter species.

Homobarrelenones: Versatile Building Blocks for the Construction of Novel and Structurally Diverse Scaffolds

AbstractWe describe the derivatization of tropolone–based homobarrelenones through simple transformations, including catalytic hydrogenations, base‐catalyzed solvolysis, and hydride reductions. Despite being structurally complex and highly functionalized, the novel compounds had no significant cytotoxicity in sperm motility assays, underscoring the importance of Michael acceptor moieties in the biological activity of this class of compounds.

Native Medicinal Plants and Their Effects on Anti-biofilm and Anti-quorum Sensing Activity

Background: Pseudomonas aeruginosa is an opportunistic pathogen with a high mortality rate, particularly among immunocompromised patients. The increase in antibiotic resistance in P. aeruginosa isolates from clinical samples has prompted researchers to seek alternative ways to reduce the pathogenicity of this microorganism. Targeting quorum sensing (QS) presents a promising approach for limiting infections caused by this bacterium. Objectives: This study aimed to evaluate mechanisms for reducing QS and controlling bacterial growth using various medicinal plants. Methods: In this study, the anti-QS effects of seven plant extracts—thyme (Thymus vulgaris), chamomile (Matricaria chamomilla), sumac (Rhus coriaria), ginger (Zingiber officinale), lemon balm (Melissa officinalis), rosehip (Echinacea angustifolia), and marshmallow (Althaea officinalis)—were investigated against P. aeruginosa. A total of 180 P. aeruginosa clinical isolates were examined. High-performance liquid chromatography (HPLC) was then used to detect phenolic compounds and flavonoids in the seven extracts. The presence of quorum-sensing genes (lasR, rhlR, and rhlI) was also evaluated. In addition, pyocyanin quantification, swarming motility assays, and biofilm formation assays were conducted with the herbal extracts. Results: The HPLC analysis results were determined based on relative times and peak areas, revealing specific peaks at different points. Molecular methods confirmed the presence of QS genes, including LasI (54%), lasR (34%), rhlR (12%), and rhlI (4%), which were used in anti-QS screening. Expression of the LasI gene decreased in P. aeruginosa strains treated with herbal extracts compared to untreated strains. Notably, Thymus vulgaris extract demonstrated the strongest inhibition, reducing violacein production by 70.5%, suppressing pyocyanin by 94%, and inhibiting swarming motility and biofilm formation by 80.3%. Conclusions: This study demonstrated that herbal extracts influence virulence factors and gene expression, particularly targeting the las gene, in P. aeruginosa isolates. We recommend further research on the extraction and identification of active ingredients, which may hold potential as therapeutic agents.

Dissecting the pH Sensitivity of Kinesin-Driven Transport.

Kinesin-1 is a crucial motor protein that drives the microtubule-based movement of organelles, vital for cellular function and health. Mostly studied at pH 6.9, it moves at approximately 800 nm/s, covers about 1 μm before detaching, and hydrolyzes one ATP per 8 nm step. Given that cellular pH is dynamic and alterations in pH have significant implications for disease, understanding how kinesin-1 functions across different pH levels is crucial. To explore this, we executed single-molecule motility assays paired with precise optical trapping techniques over a pH range of 5.5-9.8. Our results show a consistent positive relationship between increasing pH and the enhanced detachment (off rate) and speed of kinesin-1. Measurements of the nucleotide-dependent off rate show that kinesin-1 exhibits the highest rate of ATPase activity at alkaline pH, while it demonstrates the optimal number of ATP turnover and cargo translocation efficiency at the acidic pH. Physiological pH of 6.9 optimally balances the biophysical activity of kinesin-1, potentially allowing it to function effectively across a range of pH levels. These insights emphasize the crucial role of pH homeostasis in cellular function, highlighting its importance for the precise regulation of motor proteins and efficient intracellular transport.

The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

BackgroundPlesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors.ResultsIn this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides’ capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides.ConclusionsOverall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

IMMU-25. B-CELL BASED THERAPY (BVAX) PRODUCES ANTIBODIES TO SUPPRESS GLIOBLASTOMA TUMOR MIGRATION AND INVASION

Abstract Glioblastoma (GBM) presents as a highly aggressive and malignant brain tumor with limited treatment options. Despite the availability of existing therapies, the invasive characteristics of GBM frequently result in tumor recurrence and poor prognosis. We previously developed a B-cell-based therapy (BVax) that has shown promising results in its ability to induce therapeutic responses in preclinical models of GBM. Our research revealed potency of BVax can be attributed to its robust antigen-presenting capability, cognate antigen specificity with T cells, and ability to generate tumor-reactive antibodies. The present study aims to characterize the unique antigenic reactivity of BVax-derived antibodies by evaluating their immunological effects and therapeutic potential in both murine models and GBM patient-specific samples. Leveraging CD45.1 and CD45.2 congenic mouse models, we first revealed that BVax preferentially migrated to glioma-bearing brains upon intravenous injection, resulting in a CD38+CD20-CD19+ plasmablast phenotype. To further characterize the reactivity of BVax-derived immunoglobulins (Ig), we utilized proteomic analysis and identified preferential targets of Gelsolin, Fibronectin, Versican, Fibrinogen, and collagen type IV alpha, commonly associated with cell motility and the extracellular matrix. Microdialysis data confirmed the increased presence of many of these BVax-derived Igs-recognized antigens in tumor regions compared to normal brain regions in GBM patients. Utilizing purified BVax-derived antibodies from patient specimen, in vitro cell motility assays demonstrated a significant prevention of the invasion and migration of cell lines from patient derived xenograft (PDX). In this study, we concluded that BVax elicits antitumor activity through targeted migration to tumor-bearing regions, differentiation into plasmablasts, and robust secretion of specific Igs that retain the ability to inhibit functionalities associated with cell invasion and migration. Here, we present the first in-depth study that characterizes GBM patients’ antibody reactivity, specifically a subset of inflammatory B cells (BVax) with therapeutic effects that offer an indication for a new direction of immunotherapies.

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Cyclobutrifluram.

Cyclobutrifluram, a succinate dehydrogenase inhibitor fungicide, is being evaluated as a seed-applied nematicide in cotton and soybean to manage plant-parasitic nematodes. Currently, there is no information on the toxicity, ovicidal activity, nematode recovery, or effects on nematode infection for Meloidogyne incognita or Rotylenchulus reniformis after exposure to low concentrations of cyclobutrifluram. Nematode toxicity assays were performed in aqueous solutions of cyclobutrifluram, and root infection assays were conducted on tomato. Nematode paralysis was observed after 2 h of exposure to 0.5 μg/ml cyclobutrifluram for both nematode species. Based on an assay of nematode motility, the 2-h effective concentration of fungicide required for 50% growth inhibition (EC50) value for M. incognita and R. reniformis was 0.48 and 1.07 μg/ml, respectively. In a comparable assay with a similar nematicide, continuous exposure to 0.5 μg/ml cyclobutrifluram for 24 h resulted in at least 45% more immotile nematodes for both species compared with those treated with 0.5 μg/ml fluopyram. Continuous exposure to concentrations greater than 1.0 μg/ml suppressed hatching for both species compared with the water control. Nematode recovery from paralysis was greater than 80% for M. incognita and R. reniformis 24 h after nematodes were rinsed and removed from a 1-h treatment to their respective 2-h EC50 concentrations. Nematode infection of tomato roots was reduced following a 1-h treatment with aqueous solutions of cyclobutrifluram, ranging from 0.12 to 0.48 μg/ml for M. incognita and 0.27 to 1.07 μg/ml for R. reniformis. Overall, the toxicity of cyclobutrifluram to these nematode species was greater than that of fluopyram, and although the effects of cyclobutrifluram were reversible, low concentrations were effective at reducing the ability of both nematodes to infect tomato roots.

In vivo evaluation of anti-diarrheal activity of hydroalcoholic extract and solvent fractions of the leaf of Leucas deflexa Hook.f (Lamiaceae) in rodents

The emergence of antibiotic-resistant bacteria has intensified the search for novel antidiarrheal drug. Plant-derived extracts offer promising alternatives due to their ability to modulate gut motility and enhance water absorption. The leaves of Leucas deflexa Hook.f., a plant native to Ethiopia, have been traditionally utilized in the treatment of diarrheal diseases. Nonetheless, its traditional application has yet to be scientifically confirmed. Hence, the aim of the present study was to evaluate antidiarrheal activity of the crude extract and fractions of the leaf of Leucas deflexa Hook.f (Lamiaceae). Healthy Swiss albino mice were randomly divided into five groups per experimental model: castor oil-induced diarrhea, gastrointestinal motility, and anti-enteropooling assays. The control group received distilled water (10ml/kg), while the positive control was treated with loperamide (3mg/kg). The remaining groups were administered various dosages (LD100, LD200, and LD400 mg/kg) of a hydroalcoholic extract or its fractions from Leucas deflexa Hook.f. The crude extract and chloroform fraction substantially (P<0.001) delayed the onset of diarrhea, reduced fecal frequency, and decreased total fecal weight at all administered doses compared to the negative control. The butanol and aqueous fractions exhibited a substantial antidiarrheal effect at doses of 200mg/kg and 400mg/kg, respectively. All treatments, including the crude extract, chloroform, butanol, and aqueous fractions, substantially (P<0.001) reduced the volume and weight of intestinal contents at various doses. Additionally, the chloroform, aqueous fraction, and crude extract at doses of 100, 200, and 400mg/kg substantially inhibited gastrointestinal motility compared to the negative control. The results of this study corroborate the traditional use of Leucas deflexa leaves in the treatment of diarrhea. These findings give a scientific justification for further exploration of the underlying mechanisms and potential therapeutic applications of this plant extract.

Protective Effects of Nettle Tea on SKOV-3 Ovarian Cancer Cells Through ROS Production, Apoptosis Induction, and Motility Inhibition Without Altering Autophagy.

Urtica dioica L. (UD), also known as the stinging nettle, has long been used in traditional medicine for its wide range of health benefits. The current study focuses on the effect of nettle tea on the growth and proliferation of one of the most aggressive ovarian adenocarcinoma cell line, SKOV-3 cells. To examine this, cytotoxicity, cell cycle analysis, and ROS assays were performed, along with Annexin V/PI dual staining, cell death ELISA, Western blot analysis, and motility assays. The results showed that a UD aqueous extract (UDAE) can inhibit the growth and proliferation of SKOV-3 cells in a dose- and time-dependent manner by promoting cellular fragmentation. This was accompanied by an increase in two apoptotic hallmarks, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation as revealed by cell death ELISA. This aqueous extract showed a pro-oxidant activity while also activating the extrinsic caspase-dependent apoptotic pathway with no alteration in autophagy markers. Furthermore, the extract showed promising inhibitory effect on the migratory capacities of aggressive ovarian cancer cells, in vitro.

The D75N and P161S Mutations in the C0-C2 Fragment of cMyBP-C Associated with Hypertrophic Cardiomyopathy Disturb the Thin Filament Activation, Nucleotide Exchange in Myosin, and Actin-Myosin Interaction.

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

Impact of acidic and alkaline conditions on Staphylococcus aureus and Acinetobacter baumannii interactions and their biofilms.

Bacterial biofilms pose significant challenges due to their association with antibiotic resistance, metabolic adaptation, and survival under harsh conditions. Among notable pathogens forming biofilms, Staphylococcus aureus and Acinetobacter baumannii are concerning pathogens in nosocomial settings. However, their behaviour under acidic (pH 4.5) and alkaline (pH10.5) conditions, especially in co-culture setups, remains insufficiently understood. This study investigates these aspects, by examining growth rates, biofilm formation, pH shifts, phenotypic analysis, and gene expression profiles. The results showed A. baumannii exhibited reduced growth and biofilm formation at pH 4.5, while S. aureus showed slow growth and low biofilm formation at pH10.5 in mono-cultures. S. aureus leaned towards an acidic pH (6-6.5), whereas A. baumannii shifted towards an alkaline pH (8-9). In co-culture environments, growth rates and biofilm formation increased across all pH conditions, converging towards a neutral pH over time. Phenotypic motility assays indicated that A. baumannii exhibited greater motility in alkaline conditions, while S. aureus showed increased staphyloxanthin production under acidic conditions. Gene expression analyses revealed that the fibronectin-binding protein A (FnbA) and N-acetylglucosaminyl-transferase (icaA) genes, responsible for initial attachment during biofilm formation, were highly expressed in acidic co-culture condition but poorly expressed in alkaline condition. In A. baumannii, the outer membrane protein A (OmpA) gene associated with adhesion and virulence, was upregulated in co-culture. TheLuxR gene involved in quorum sensing was upregulated in acidic conditions and poorly expressed at pH 10.5. This study elucidates the metabolic adaptability and biofilm formation tendencies of S. aureus towards acidic conditions and A. baumannii towards alkaline conditions, providing insights for better management of biofilm-related infections.

Improved longevity of actomyosin in vitro motility assays for sustainable lab-on-a-chip applications

AbstractIn the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21–26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21–26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Prognostic Significance and Immune Landscape of an Efferocytosis-Related Gene Signature in Bladder Cancer.

Bladder cancer poses a significant global health challenge, underscoring the imperative for precise prognostic instruments to advance patient care. Against the backdrop of efferocytosis's increasingly recognized role in cancer, this research endeavors to develop and authenticate a prognostic signature intricately linked to efferocytosis in bladder cancer. LASSO-COX regression analysis crafted an efferocytosis-related genes risk prognostic model, followed by the construction of a column chart. External validation sets confirmed the predictive accuracy of both the model and chart. Clinical, tumor microenvironment, drug sensitivity, and immunotherapy analyses were employed to comprehensively assess efferocytosis-related scores. The expression of TGFB3 key genes was validated via RT-PCR and western blotting. Further validation included Transwell, Wound healing, Colony formation, and EDU assays. We formulated and validated an efferocytosis-related genes risk model in bladder cancer, comprising 13 core genes. The risk model demonstrated autonomous prognostic significance in both univariate and multivariate Cox analyses. Following the multivariate analysis, we devised a nomogram. Moreover, by utilizing individual risk scores derived from this risk model, we successfully stratified patients into two discernible risk cohorts, unveiling noteworthy variances in immune infiltration profiles and responsiveness to immunotherapy. Notably, the model's key gene TGFB3 was validated through comprehensive experimental investigations, including Transwell assays for migration and invasion and Wound healing assays for motility on the T24 and BIU cell lines. This study has furnished innovative perspectives on an efferocytosis-related prognostic signature, elucidating the prognosis and immune milieu intricacies in patients with bladder cancer.

Impact of Quorum Sensing on the Virulence and Survival Traits of Burkholderia plantarii.

Quorum sensing (QS) is a mechanism by which bacteria detect and respond to cell density, regulating collective behaviors. Burkholderia plantarii, the causal agent of rice seedling blight, employs the LuxIR-type QS system, common among Gram-negative bacteria, where LuxI-type synthase produces QS signals recognized by LuxR-type regulators to control gene expression. This study aimed to elucidate the QS mechanism in B. plantarii KACC18965. Through whole-genome analysis and autoinducer assays, the plaI gene, responsible for QS signal production, was identified. Motility assays confirmed that C8-homoserine lactone (C8-HSL) serves as the QS signal. Physiological experiments revealed that the QS-defective mutant exhibited reduced virulence, impaired swarming motility, and delayed biofilm formation compared to the wild type. Additionally, the QS mutant demonstrated weakened antibacterial activity against Escherichia coli and decreased phosphate solubilization. These findings indicate that QS in B. plantarii significantly influences various pathogenicity and survival traits, including motility, biofilm formation, antibacterial activity, and nutrient acquisition, highlighting the critical role of QS in pathogen virulence and adaptability.

In vitro anthelmintic activity of hydroethanolic extract of Artemisia herba aba and Juniperus phoenicea on abomasal Teladorsagia circumcincta of sheep.

The present study aimed to evaluate the in vitro anthelmintic activity of Juniperus phoenicea and Artemisia herba-alba against abomasal nematodes collected from naturally infected sheep. The anthelmintic activities of the extracts were assessed using egg hatch inhibition (EHIA) and adult motility assays (AMA) against Teladorsagia circumcincta. The parasites were stored in phosphate-buffered saline (PBS) until the in vitro assessment began. The leaf powders of J. phoenicea and A. herba-alba were extracted using the maceration procedure. Briefly, the leaf powders were mixed with a sufficient quantity of 99% ethanol and distilled water (80%/20%). The extracts were dissolved in a 3% dimethyl sulfoxide (DMSO) solution to improve water solubility. The hydroethanolic extracts were tested at concentrations of 1.562, 3.125, 6.25, 12.5, and 25 mg/ml. Adult motile worms were individually subjected to each treatment at laboratory temperature. The PBS and albendazole (25 mg/ml) were prepared and used as negative and positive controls, respectively. The test was repeated five times for each concentration. The results showed that both plant extracts exhibited anthelmintic effects. A. herba-alba extract at 25 mg/ml inhibited the motility of all T. circumcincta adult worms within 1 hour of administration, while J. phoenicea extract caused 100% mortality of adult worms after 4 hours at the same concentration. Neither of the extracts, at 1.562 mg/ml, had any inhibitory effect on motility. At 25 mg/ml, the A. herba-alba (98.67%) and J. phoenicea (96.24%) showed high activity with an inhibitory effect on hatchability compared to the other tested concentrations. The findings of the present study showed that hydroethanolic extracts of A. herba-alba and J. phoenicea leaves had in vitro anthelmintic activity on eggs and adult T. circumcincta of sheep.

Extracts and Terpenoids from Stevia Species as Potential Anthelmintics for Neglected Tropical Diseases Caused by Cestode Parasites.

Cestodes are etiological agents of neglected diseases such as echinococcosis and cysticercosis, which are major public health problems. Antiparasitic treatment relies on a small number of approved drugs, which are often only partially effective, poorly tolerated and require prolonged administration. Thus, the discovery of novel potential treatments is critical. The Stevia genus (Asteraceae) includes species that are recognized as a source of bioactive compounds, with many species associated with medicinal uses. In this study, the cestocidal activity of four South American Stevia species that previously showed antiprotozoal activity was analyzed using a motility assay on the laboratory cestode model, Mesocestoides vogae. The four Stevia extracts showed cestocidal activity, with S. alpina var. alpina as the most active. The sesquiterpene lactones estafietin and eupatoriopicrin were purified from S. alpina var. alpina and S. maimarensis, respectively, and tested on M. vogae. Estafietin showed cestocidal activity, inhibiting parasite viability in a dose-dependent manner, even from the first day of incubation. Consistent with the motility effects, the extract of S. alpina var. alpina and estafietin induced marked alterations in the morphology of the parasite. The results of this report show that Stevia species represent a source of new molecules with potential for the treatment of neglected tropical diseases caused by cestodes.

Characterizing human KIF1Bβ motor activity by single-molecule motility assays and Caenorhabditis elegans genetics.

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1Bβ is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1Bβ lead to a wide range of neuropathies. Although previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1Bβ remain elusive. In this study, we determined the motile parameters of KIF1Bβ through single-molecule motility assays. We found that the C-terminal region of KIF1Bβ has an inhibitory role in motor activity. AlphaFold2 prediction suggests that the C-terminal region blocks the motor domain. Additionally, we established simple methods for testing the axonal transport activity of human KIF1Bβ using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1Bβ activities, both in vitro and in vivo, caused by a Charcot-Marie-Tooth disease-associated Q98L mutation.

Myosin-I synergizes with Arp2/3 complex to enhance the pushing forces of branched actin networks.

Class I myosins (myosin-Is) colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by the Arp2/3 complex on the surface of beads coated with myosin-I and nucleation-promoting factor. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Myosin-I triggered symmetry breaking and comet tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations, suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.