Motility Assay Research Articles

Optimizing the xWORM assay for monitoring hookworm larvae motility

Parasitic worms (helminths) infect almost all taxa across the animal kingdom, and pose significant challenges to public health and economies, particularly in developing countries. To address this problem, researchers have developed various tools to measure the motility and viability of helminths. However, the conditions used in anthelmintic screening assays are often not optimized, and can vary considerably between research teams. These unoptimized conditions may impact novel drug screens, as little is known about the effects of different conditions on the health of the target parasites. To improve future research, this study determined the effects of key assay parameters including, media type, media concentration, in-well parasite density, and assay duration on the infective third-stage larva (L3) of two types of hookworms, namely Nippostrongylus brasiliensis in rodents, and Necator americanus in humans. Conditions were screened over several days using the xCELLigence worm real-time motility assay (xWORM); a real-time impedance-based helminth motility assay using the xCELLigence system with 96-well microplates. While results varied depending on the species and media used, the study found that 500–1,000 L3/200-µL and a media concentration of 3.13–25% generally produced good to excellent assay conditions. The findings of this study can guide the future selection of xWORM assay parameters for novel drug trials involving these parasite species and serve as a suggested model for optimizing trial conditions for alternative parasite targets and assays.

Anthelmintic Activity of Methanolic Extract of Leaves Of Cyclea Peltata Lam and it’s Statistical Analysis

The aim of this research is to develop a safe and effective anthelmintic from plants, due to the resistance and toxic effect of synthetic anthelmintics on animals and humans. There are several side effects for synthetic drugs including drug resistance, toxicity, and drug residual in animal products. So there is a need for the exploration of medicinal plants for the treatment of different types of worm infections.
 In this research a medicinally and traditionally important plant is used, leaves of Cyclea peltata Lam. The methanolic extract of the leaves were prepared by maceration technique and are monitored for the anti-helminthic activity against adult Pheretima posthuma worms. The reason for selecting this plant is due to the presence of tannins, which have been proved by previous phytochemical studies. Tannins have the capability to bind free proteins in the cuticle, oral cavity of helminths, thus causing the death. Albendazole (25 mg/5ml) is used as a controlled drug by using the adult motility assay method. Three concentrations were prepared (25, 50, and 100mg/5ml). The time required for the immobility and death of the worms were determined. This study deals with the statistical analysis by one way ANOVA followed by Tukey’s multiple comparison test at 95% confidence interval using graph pad prism software version 9.0.0 reveals the result that the methanolic extract of the Leaves of Cyclea peltata Lam causes the immobility and death of the vermicular in a concentration-dependent manner as compared to the standard drug Albendazole.

Qualified sperm selection based on the rheotaxis and thigmotaxis in a microfluidic system.

In the present study, we used a microfluidic system for studying the combined effects of thigmotaxis and rheotaxis for sperm separation process to achieve the successful Assisted reproductive technology (ART). The designed PDMS-based microfluidic system had four various geometries, including linear, square, zigzag, and sinusoidal. The functionality of separated sperm was evaluated by sperm tracking (ImageJ), motility assay (CASA software), and morphology assay (Papanicolaou ultrafast staining). Probing various geometries revealed 100% motility. In non-linear geometries, sperm's periodic detachment from the walls gave rise to the periodic interaction with the high flow velocity in the center of the channel, resulting in the separation of high-quality sperm with progressive motility. The collected data proved the influence of thigmotaxis on the quality of separated sperm. Morphologically improvement in separated sperm from the sinusoidal geometry was significant than others, which means the sinusoidal structure would be the best candidate for the sperm separation process. The online version contains supplementary material available at 10.1007/s13534-023-00294-8.

Rhoptry neck protein 4 plays important roles during Plasmodium sporozoite infection of the mammalian liver.

During invasion, Plasmodium parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver in vivo. Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, Plasmodium berghei, we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver in vivo. Combined with in vitro investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in Plasmodium merozoites and Toxoplasma tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of Apicomplexa parasites.

The role of ADAM8 in the mechanophenotype of MDA-MB-231 breast cancer cells in 3D extracellular matrices.

The majority of investigations of cancer cells have been performed in an oversimplified 2D in vitro environment. In the last decade there is a trend toward more sophisticated 3D in vitro cell culture model systems that can bridge the existing gap between 2D in vitro and in vivo experiments in the field of biophysical and cell biological cancer cell research. Here, we hypothesize that the bidirectional interplay between breast cancer cells and their tumor microenvironment is critical for the outcome of the disease. Thereby, the tissue remodeling processes evoked by cancer cells are important for cancer cell-driven mechanical probing of their matrix environment and on cancer cell adhesion and motility. When remodeling processes have been explored, the emphasis was placed on matrix metalloproteinases and rather not on a disintegrin and metalloproteases (ADAMs). However, the role of ADAM8 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. Thus, in this study, we focus on the function of ADAM8 in matrix remodeling and migration of 3D extracellular matrix scaffolds. Therefore, human MDA-MB-231 breast carcinoma cells with ADAM8 knocked down, referred to as ADAM8-KD cells, as well as MDA-MB-231 scrambled control cells, referred to as ADAM8-Ctrl cells, have been used to examine their ability to interact with and migrate in dense extracellular 3D matrices. The fiber displacements, as the capacity of cells to deform the environmental 3D matrix scaffold, has been observed. ADAM8-KD cells displace collagen fibers more strongly than ADAM8-Ctrl cells. Moreover, ADAM8-KD cells migrated more numerous in 3D collagen matrices compared to ADAM8-Ctrl cells. The impairment of ADAM8 using the ADAM8 inhibitor BK-1361 led to significantly increased fiber displacements of ADAM8-Ctrl cells to the levels of ADAM8-KD cells. In contrast, the inhibitor had no effect on ADAM8-KD cells in terms of fiber displacements as well as on the quantitative characteristics of cell invasion of ADAM8-Ctrl cells, albeit the cells that were found in the matrix invaded considerably deeper. When matrix remodeling by cells is impaired through GM6001, a broad-band metalloproteinase inhibitor, the fiber displacements of both cell types increased. In fact, ADAM8 is known to degrade fibronectin in a direct and/or indirect manner. The supplementation of fibronectin before polymerization of the 3D collagen matrices caused an enhancement in fiber displacements as well as in cell invasion into fibronectin-collagen matrices of ADAM8-Ctrl cells, whereas the fiber displacements of ADAM8-KD cells did not change. However, fibrinogen and laminin supplementation induced an increase in fiber displacements of both cell types. Thus, the impact of fibronectin on selective increase in fiber displacement of ADAM8-Ctrl cells appears to be ADAM8-dependent. As a consequence, the presence of ADAM8 may provide an explanation for the longstanding controversial results of fibronectin enrichment on malignant progression of cancers such as breast cancer. Finally, ADAM8 is apparently essential for providing cell-driven fiber displacements of the extracellular matrix microenvironment, which fosters 3D motility in a fibronectin-rich environment. Contribution to the field. Currently, the role of ADAM8 has been explored in 2D or at maximum 2.5D in vitro cell culture motility assays. However, the mechanical characteristics of these two cell types have not been examined. In this study, the function of ADAM8 in breast cancer is refined by providing in vitro cell investigations in 3D collagen fiber matrices of various conditions. ADAM8 has been shown to be involved in the reduced generation of fiber displacements and in influencing breast cancer cell migration. However, especially in the presence of fibronectin in 3Dcollagen fiber matrices, the fiber displacements of ADAM8-Ctrl cells are increased.

Broad-Spectrum Inhibitors for Conserved Unique Phosphoethanolamine Methyltransferases in Parasitic Nematodes Possess Anthelmintic Efficacy.

In humans, nematode infections are prevalent in developing countries, causing long-term ill health, particularly in children. Worldwide, nematode infections are prevalent in livestock and pets, affecting productivity and health. Anthelmintic drugs are the primary means of controlling nematodes, but there is now high prevalence of anthelmintic resistance, requiring urgent identification of new molecular targets for anthelmintics with novel mechanisms of action. Here, we identified orthologous genes for phosphoethanolamine methyltransferases (PMTs) in nematodes within the families Trichostrongylidae, Dictyocaulidae, Chabertiidae, Ancylostomatoidea, and Ascarididae. We characterized these putative PMTs and found that they possess bona fide PMT catalytic activities. By complementing a mutant yeast strain lacking the ability to synthesize phosphatidylcholine, the PMTs were validated to catalyze the biosynthesis of phosphatidylcholine. Using an in vitro phosphoethanolamine methyltransferase assay with PMTs as enzymes, we identified compounds with cross-inhibitory effects against the PMTs. Corroboratively, treatment of PMT-complemented yeast with the PMT inhibitors blocked growth of the yeast, underscoring the essential role of the PMTs in phosphatidylcholine synthesis. Fifteen of the inhibitors with the highest activity against complemented yeast were tested against Haemonchus contortus using larval development and motility assays. Among them, four were found to possess potent anthelmintic activity against both multiple drug-resistant and susceptible isolates of H. contortus, with IC50 values (95% confidence interval) of 4.30 μM (2.15-8.28), 4.46 μM (3.22-6.16), 28.7 μM (17.3-49.5), and 0.65 μM (0.21-1.88). Taken together, we have validated a molecular target conserved in a broad range of nematodes and identified its inhibitors that possess potent in vitro anthelmintic activity.

Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points.

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM), and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT, and 2mM BHT). Semen samples were put into French straws (0.25mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future.

Microtubule detyrosination drives symmetry breaking to polarize cells for directed cell migration

To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.

The chaperone ClpC participates in sporulation, motility, biofilm, and toxin production of Clostridioides difficile

In this study, we analyzed the role of ClpC chaperone-protease of C. difficile in virulence-related traits by comparing the phenotypes of a wild-type and a mutant strain lacking the clpC gene (ΔclpC). We performed biofilm, motility, spore formation, and cytotoxicity assays. Our results show significant differences between the wild-type and ΔclpC strains in all analyzed parameters. Based on these findings, we conclude that clpC plays a role in the virulence properties of C. difficile.

The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X.

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera

BackgroundAmyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43.MethodsBy applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.ResultsFour PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system.ConclusionsOur study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Influences of flavonoids from Sedum aizoon L. on biofilm formation of Pseudomonas fragi.

Pseudomonas fragi (P. fragi) is one of the main categories of bacteria responsible for the spoilage of chilled meat. In the processing and preservation of chilled meat, it is easy to form biofilms on the meat, leading to the development of slime on the meat, which becomes a major quality defect. Flavonoids, as one of the critical components of secondary plant metabolites, are receiving increasing attention for their antibacterial activity. Flavonoids in Sedum aizoon L. (FSAL), relying on its prominent antibacterial activity, are of research importance in food preservation and other applications. This article aims to investigate the effect of FSAL on the biofilm formation of P. fragi, to better apply FSAL to the processing and preservation of meat products. The disruption of cellular structure and aggregation properties by FSAL was demonstrated by the observation of the cellular state within the biofilm. The amount of biofilm formation was determined by crystal violet staining, and the content of polysaccharides and proteins in the extracellular wrapped material was determined. It was shown that the experimental concentrations of FSAL (1.0 MIC) was able to inhibit biofilm formation and reduce the main components in the extracellular secretion. The swimming motility assay and the downregulation of flagellin-related genes confirmed that FSAL reduced cell motility and adhesion. The downregulation of cell division genes and the lowering of bacterial metabolic activity suggested that FSAL could hinder bacterial growth and reproduction within P. fragi biofilms. KEY POINTS: • FSAL inhibited the activity of Pseudomonas fragi in the dominant meat strain • The absence of EPS components affected the formation of P. fragi biofilms • P. fragi has reduced adhesion capacity due to impaired flagellin function.

Horizontal Magnetic Tweezers to Directly Measure the Force-Velocity Relationship for Multiple Kinesin Motors.

Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities whichweattribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.

DegS protease regulates the motility, chemotaxis, and colonization of Vibrio cholerae.

In bacteria, DegS protease functions as an activating factor of the σE envelope stress response system, which ultimately activates the transcription of stress response genes in the cytoplasm. On the basis of high-throughput RNA sequencing, we have previously found that degS knockout inhibits the expression of flagellum synthesis- and chemotaxis-related genes, thereby indicating that DegS may be involved in the regulation of V. cholerae motility. In this study, we examined the relationships between DegS and motility in V. cholerae. Swimming motility and chemotaxis assays revealed that degS or rpoE deletion promotes a substantial reduction in the motility and chemotaxis of V. cholerae, whereas these activities were restored in ΔdegS::degS and ΔdegSΔrseA strains, indicating that DegS is partially dependent on σE to positively regulate V. cholerae activity. Gene-act network analysis revealed that the cAMP-CRP-RpoS signaling pathway, which plays an important role in flagellar synthesis, is significantly inhibited in ΔdegS mutants, whereas in response to the overexpression of cyaA/crp and rpoS in the ΔdegS strain, the motility and chemotaxis of the ΔdegS + cyaA/crp and ΔdegS + rpoS strains were partially restored compared with the ΔdegS strain. We further demonstrated that transcription levels of the flagellar regulatory gene flhF are regulated by DegS via the cAMP-CRP-RpoS signaling pathway. Overexpression of the flhF gene in the ΔdegS strain partially restored motility and chemotaxis. In addition, suckling mouse intestinal colonization experiments indicated that the ΔdegS and ΔrpoE strains were characterized by the poor colonization of mouse intestines, whereas colonization efficacy was restored in the ΔdegSΔrseA, ΔdegS + cyaA/crp, ΔdegS + rpoS, and ΔdegS + flhF strains. Collectively, our findings indicate that DegS regulates the motility and chemotaxis of V. cholerae via the cAMP-CRP-RpoS-FlhF pathway, thereby influencing the colonization of suckling mouse intestines.

A novel purgative mechanism of multiflorin A involves changing intestinal glucose absorption and permeability

BackgroundMultiflorin A (MA) is a potential active ingredient of traditional herbal laxative, Pruni semen, with unusual purgative activity and an unclear mechanism, and inhibiting intestinal glucose absorption is a promising mechanism of novel laxatives. However, this mechanism still lacks support and a description of basic research. PurposeThis study aimed to determine the main contribution of MA to the purgative activity of Pruni semen and elucidate the effect intensity, characteristics, site, and mechanism of MA in mice, and determine the novel mechanism of traditional herbal laxatives from the perspective of intestinal glucose absorption. MethodsWe induced diarrhoea in mice by administering Pruni semen and MA, and the defecation behaviour, glucose tolerance, and intestinal metabolism were analysed. The effects of MA and its metabolite on peristalsis of the intestinal smooth muscle were evaluated using an intestinal motility assay in vitro. Intestinal tight junction proteins, aquaporins, and glucose transporters expression were analysed using immunofluorescence; gut microbiota and faecal metabolites were analysed using 16S rRNA and liquid chromatography-mass spectrometry. ResultsMA administration (20 mg/kg) induced watery diarrhoea in over half of the experimental mice. The activity of MA in lowering peak postprandial glucose levels was synchronous with purgative action, with the acetyl group being the active moiety. MA was metabolised primarily in the small intestine, where it decreased sodium-glucose cotransporter-1, occludin, and claudin1 expression, then inhibited glucose absorption, resulting in a hyperosmotic environment. MA also increased the aquaporin3 expression to promote water secretion. Unabsorbed glucose reshapes the gut microbiota and their metabolism in the large intestine and the increasing gas and organic acid promoted defecation. After recovery, the intestinal permeability and glucose absorption function returned, and the abundance of probiotics such as Bifidobacterium increased. ConclusionThe purgative mechanism of MA involves inhibiting glucose absorption, altering permeability and water channels to promote water secretion in the small intestine, and regulating gut microbiota metabolism in the large intestine. This study is the first systematic experimental study on the purgative effect of MA. Our findings provide new insight into the study of novel purgative mechanisms.

Characterization and antivibrio activity of chitosan-citral Schiff base calcium complex for a calcium citrate sustained release antibacterial agent

Vibrio parahemolyticus is the “Number one killer” of seafood products. Anti-vibrio agents having low cost and high-safety are urgently needed to supplement the application needs. This work attempted to prepare CS-CT-CCa complex with citral (CT), chitosan (CS) and calcium citrate (CCa) as raw material by microwave-assisted high-pressure homogenization. Additionally the coordination structure and morphology of Bridge-CS-CT-Schiff base/OH-CCa were verified. The prepared CS-CT-CCa had a well-dispersed property (the size: 3.55~9.33 μm and the zeta potential: +38.7~+67.5 mV) and an excellent sustained released ability (sustained release up to 180 min). MIC, Glucose assay, MDA assay, biofilm formation inhibition assay, SEM, swimming and swarming motility assay demonstrated that CS-CT-CCa had strong (MIC of 128 μg/mL) and sustained (more than 12 h) inhibitory effects against V. parahaemolyticus. Meanwhile, CS-CT-CCa could increase the membrane permeability of V. parahaemolyticus and inhibit their biofilm-forming ability in a dose-dependent manner. It could be inferred that the antibacterial activities against V. parahaemolyticus caused inhibition of biofilm formation, swimming and swarming motilities. This study provided necessary data for the further design and development of chitosan antibacterial agents, food and feed additives.

Abstract 2465: YKL-40 overexpression enhances metastatic potential and is a novel prognostic candidate and predictive biomarker for patients with colorectal cancer

Abstract Purpose: Although early detection and high-quality treatment options, metastatic colorectal cancer (mCRC) is the main cause of CRC-related mortality. Dissecting the mechanisms underlying CRC progression may identify new accurate markers for invasive tumors, which can also be used as druggable targets, providing further improvement in the patients clinical outcomes. YKL-40 is a glycosyl hydrolase functionally linked to increased proliferation, angiogenesis, migration, and invasion of tumor cells. Because in colorectal cancer (CRC), YKL-40 serological level may serve as a risk predictor and prognostic biomarker, we investigated the underlying mechanisms by which it may contribute to tumor progression and the clinical significance of its tissue expression in metastatic CRC. Experimental Design: HCT116 and Caco2 cells genetically engineered to achieve YKL-40 silencing/overexpression were used for cell motility, invasion and proliferation assays, and to measure EMT markers. The YKL-40 expression level was assessed in the early and late tumor phases obtained in the AOM/DSS mouse model, as well as in tumors and sera from CRC patients. Six independent cohorts of CRC patients were stratified by YKL-40 tissue expression to define its prognostic role and its effect on cetuximab and oxaliplatin treatment response. Results: YKL-40 high-expressing HCT116 and Caco2 cells showed increased motility, invasion and proliferation. YKL-40 up-regulation was associated with EMT signaling activation. In murine and human samples, elevated YKL-40 levels correlated with high-grade tumors. In retrospective analyses, YKL-40 elevated expression correlated with shorter survival in patients with advanced CRC. Strikingly, YKL-40 high tissue levels showed a predictive value for better response to cetuximab, even in patients with stage IV CRC and mutant KRAS, and worse sensitivity to oxaliplatin. Conclusions: Our study recognizes a novel role of YKL-40 tissue expression in promoting CRC metastatic potential, through EMT signaling activation, and provides significant clinical implications that may impact the risk prediction of patients with mCRC. YKL-40 high tissue levels also strengthen a predictive value for better cetuximab responsiveness, even in patients with KRAS mutations. Since resistance to cetuximab remains one of the greatest challenges in treating CRC, our findings may be crucial for developing novel YKL-40-targeted therapy approaches. Citation Format: Mariangela De Robertis, Maria Raffaella Greco, Rosa Angela Cardone, Tommaso Mazza, Flaviana Marzano, Nikolay Mehterov, Maria Kazakova, Nikolay Belev, Apollonia Tullo, Graziano Pesole, Victoria Sarafian, Emanuela Signori. YKL-40 overexpression enhances metastatic potential and is a novel prognostic candidate and predictive biomarker for patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2465.

Paracrine relationship between incretin hormones and endogenous 5-hydroxytryptamine in the small and large intestine.

Enterochromaffin (EC) cell-derived 5-hydroxytryptamine (5-HT) is a mediator of toxin-induced reflexes, initiating emesis via vagal and central 5-HT3 receptors. The amine is also involved in gastrointestinal (GI) reflexes that are prosecretory and promotile, and recently 5-HT's roles in chemosensation in the distal bowel have been described. We set out to establish the efficacy of 5-HT signaling, local 5-HT levels and pharmacology in discrete regions of the mouse small and large intestine. We also investigated the inter-relationships between incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) and endogenous 5-HT in mucosal and motility assays. Adult mouse GI mucosae were mounted in Ussing chambers and area-specific studies were performed to establish the 5-HT3 and 5-HT4 pharmacology, the sidedness of responses, and the inter-relationships between incretins and endogenous 5-HT. Natural fecal pellet transit in vitro and full-length GI transit in vivo were also measured. We observed the greatest level of tonic and exogenous 5-HT-induced ion transport and highest levels of 5-HT in ascending colon mucosa. Here both 5-HT3 and 5-HT4 receptors were involved but elsewhere in the GI tract epithelial basolateral 5-HT4 receptors mediate 5-HT's prosecretory effect. Exendin-4 and GIP induced 5-HT release in the ascending colon, while L cell-derived PYY also contributed to GIP mucosal effects in the descending colon. Both peptides slowed colonic transit. We provide functional evidence for paracrine interplay between 5-HT, GLP-1 and GIP, particularly in the colonic mucosal region. Basolateral epithelial 5-HT4 receptors mediated both 5-HT and incretin mucosal responses in healthy colon.