Reusable mechano-bactericidal surface with echinoid-shaped hierarchical micro/nano-structure.

Biofilms formed owing to the attachment of bacteria to surfaces have caused various problems in industries such as marine transportation/logistics and medicine. In response, many studies have been conducted on bactericidal surfaces, and nanostructured surfaces mimicking cicada and dragonfly wings are emerging as candidates for mechano-bactericidal surfaces. In specific circumstances involving mechano-bactericidal activity, certain nanostructured surfaces could exhibit their bactericidal effects by directly deforming the membranes of bacteria that adhere to these nanostructures. Additionally, in most cases, debris of bacterial cells may accumulate on these nanostructured surfaces. Such accumulation poses a significant challenge: it diminishes the mechano-bactericidal effectiveness of the surface, as it hinders the direct interaction between the nanostructures and any new bacteria that attach subsequently. In specific circumstances involving mechano-bactericidal activity, certain nanostructured surfaces could exhibit their bactericidal effects by directly deforming the membranes of bacteria that adhere to these nanostructures. Additionally, in most cases, debris of bacterial cells may accumulate on these nanostructured surfaces. Such accumulation poses a significant challenge: it diminishes the mechano-bactericidal effectiveness of the surface, as it hinders the direct interaction between the nanostructures and any new bacteria that attach subsequently.In other words, there is a need for strategies to remove the accumulated bacterial debris in order to sustain the mechano-bactericidal effect of the nanostructured surface. In this study, hierarchical micro/nano-structured surface (echinoid-shaped nanotextures were formed on Al micro-particle's surfaces) was fabricated using a simple pressure-less sintering method, and effective bactericidal efficiency was shown against E. coli (97 ± 3.81%) and S. aureus (80 ± 9.34%). In addition, thermal cleaning at 500 °C effectively eliminated accumulated dead bacterial debris while maintaining the intact Al2O3 nanostructure, resulting in significant mechano-bactericidal activity (E. coli: 89 ± 6.86%, S. aureus: 75 ± 8.31%). As a result, thermal cleaning maintains the intact nanostructure and allows the continuance of the mechano-bactericidal effect. This effect was consistently maintained even after five repetitive use (E. coli: 80 ± 16.26%, S. aureus: 76 ± 12.67%).

Fabrication of Nanostructured Polycaprolactone (PCL) Film Using a Thermal Imprinting Technique and Assessment of Antibacterial Function for Its Application.

In the use of the medical devices, it is essential to prevent the attachment of bacteria to the device surface or to kill the attached bacteria. To kill bacteria, many researchers have used antibiotics or studied nanostructure-based antibacterial surfaces, which rely on mechanical antibacterial methods. Several polymers are widely used for device fabrication, one of which is polycaprolactone (PCL). PCL is biocompatible, biodegradable, easy to fabricate using 3D printing, relatively inexpensive and its quality is easily controlled; therefore, there are various approaches to its use in bio-applications. In addition, it is an FDA-approved material, so it is often used as an implantable material in the human body. However, PCL has no inherent antibacterial function, so it is necessary to develop antibacterial functions in scaffold or film-based PCL medical devices. In this study, process parameters for nanopillar fabrication were established through a simple thermal imprinting method with PCL. Finally, a PCL film with a flexible and transparent nanopillar structure was produced, and the mechano-bactericidal potential was demonstrated using only one PCL material. PCL with nanopillars showed bactericidal ability against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) bacteria cultured on its surface that resulted in membrane damage and death due to contact with nanopillars. Additionally, bacteriostatic results were shown to inhibit bacterial growth and activity of Staphylococcus aureus (S. aureus) on PCL nanostructured columns. The fabricated nanopillar structure has confirmed that mechanically induced antibacterial function and can be applied to implantable medical devices.

PEGDMA-Based Pillar-Shape Nanostructured Antibacterial Films Having Mechanical Robustness.

Antibacterial surfaces are one of the most important surfaces in the medical and marine industries. Many researchers are studying antibacterial surfaces to kill bacteria or prevent adhesions. Various materials and structures are applied to the surface to inhibit the adhesion of bacteria or kill the adhered bacteria. Nowadays, a dual strategy is preferred rather than a single strategy. In this study, nanopillar structures were fabricated using polyethylene glycol dimethacrylate (PEGDMA), which has an antifouling effect. Afterward, the fabricated nanostructured PEGDMA was assessed to confirm the intrinsic antibacterial effect and mechanically induced antibacterial functions. The adhesion of Gram-negative and Gram-positive bacteria can be effectively reduced by the PEG hydration layer formation, steric repulsion, and flexible chain, and the nanostructure can damage the bacterial membrane. In addition, we performed antibacterial experiments on a nanopillar-structured surface made of PEGDMA. Furthermore, we revealed that the mechanical robustness of the nanopillared surface was superior to that of the nanocone-structured surface using computational analysis. Nanopillar structures fabricated using PEGDMA are promising candidates for antifouling and antibacterial surfaces and can be applied in various industries.

Evaluation of the Antibacterial Activity and Cell Response for 3D-Printed Polycaprolactone/Nanohydroxyapatite Scaffold with Zinc Oxide Coating.

Among 3D-printed composite scaffolds for bone tissue engineering, researchers have been attracted to the use of zinc ions to improve the scaffold's anti-bacterial activity and prevent surgical site infection. In this study, we assumed that the concentration of zinc ions released from the scaffold will be correlated with the thickness of the zinc oxide coating on 3D-printed scaffolds. We investigated the adequate thickness of zinc oxide coating by comparing different scaffolds' characteristics, antibacterial activity, and in vitro cell response. The scaffolds' compressive modulus decreased as the zinc oxide coating thickness increased (10, 100 and 200 nm). However, the compressive modulus of scaffolds in this study were superior to those of other reported scaffolds because our scaffolds had a kagome structure and were made of composite material. In regard to the antibacterial activity and in vitro cell response, the in vitro cell proliferation on scaffolds with a zinc oxide coating was higher than that of the control scaffold. Moreover, the antibacterial activity of scaffolds with 100 or 200 nm-thick zinc oxide coating on Escherichia coli was superior to that of other scaffolds. Therefore, we concluded that the scaffold with a 100 nm-thick zinc oxide coating was the most appropriate scaffold to use as a bone-regenerating scaffold, given its mechanical property, its antibacterial activity, and its in vitro cell proliferation.

A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans.

Animals use pheromones as a conspecific chemical language to respond appropriately to environmental changes. The soil nematode Caenorhabditis elegans secretes ascaroside pheromones throughout the lifecycle, which influences entry into dauer phase in early larvae, in addition to sexual attraction and aggregation. In adult hermaphrodites, pheromone sensory signals perceived by worms usually elicit repulsion as an initial behavioral signature. However, the molecular mechanisms underlying neuronal pheromone sensory process from perception to repulsion in adult hermaphrodites remain poorly understood. Here, we show that pheromone signals perceived by GPA-3 is conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role by controlling glutaminase gene expression. We further provide evidence that this modulatory role for DAF-16/FoxO seems to be conserved evolutionarily by electro-physiological study in mouse primary hippocampal neurons that are responsible for glutamatergic neurotransmission. These findings provide the basis for understanding the nematode pheromone signaling, which seems crucial for adaptation of adult hermaphrodites to changes in environmental condition for survival.

The genetic basis of natural variation in a phoretic behavior.

Phoresy is a widespread form of commensalism that facilitates dispersal of one species through an association with a more mobile second species. Dauer larvae of the nematode Caenorhabditis elegans exhibit a phoretic behavior called nictation, which could enable interactions with animals such as isopods or snails. Here, we show that natural C. elegans isolates differ in nictation. We use quantitative behavioral assays and linkage mapping to identify a genetic locus (nict-1) that mediates the phoretic interaction with terrestrial isopods. The nict-1 locus contains a Piwi-interacting small RNA (piRNA) cluster; we observe that the Piwi Argonaute PRG-1 is involved in the regulation of nictation. Additionally, this locus underlies a trade-off between offspring production and dispersal. Variation in the nict-1 locus contributes directly to differences in association between nematodes and terrestrial isopods in a laboratory assay. In summary, the piRNA-rich nict-1 locus could define a novel mechanism underlying phoretic interactions.Nematodes use a characteristic set of movements, called nictation, to hitchhike on more mobile animals. Here, Lee et al. identify a genetic locus in the nematode Caenorhabditis elegans that underlies nictation and contributes to successful hitchhiking, but at expense of reduced offspring production.

HSF-1 is involved in regulation of ascaroside pheromone biosynthesis by heat stress in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans survives by adapting to environmental stresses such as temperature extremes by increasing the concentrations of ascaroside pheromones, termed ascarosides or daumones, which signal early C. elegans larvae to enter a non-aging dauer state for long-term survival. It is well known that production of ascarosides is stimulated by heat stress, resulting in enhanced dauer formation by which worms can adapt to environmental insults. However, the molecular mechanism by which ascaroside pheromone biosynthesis is stimulated by heat stress remains largely unknown. In the present study, we show that the heat-shock transcription factor HSF-1 can mediate enhanced ascaroside pheromone biosynthesis in response to heat stress by activating the peroxisomal fatty acid ß-oxidation genes in C. elegans. To explore the potential molecular mechanisms, we examined the four major genes involved in the ascaroside biosynthesis pathway and then quantified the changes in both the expression of these genes and ascaroside production under heat-stress conditions. The transcriptional activation of ascaroside pheromone biosynthesis genes by HSF-1 was quite notable, which is not only supported by chromatin immunoprecipitation assays, but also accompanied by the enhanced production of chemically detectable major ascarosides (e.g. daumones 1 and 3). Consequently, the dauer formation rate was significantly increased by the ascaroside pheromone extracts from N2 wild-type but not from hsf-1(sy441) mutant animals grown under heat-stress conditions. Hence heat-stress-enhanced ascaroside production appears to be mediated at least in part by HSF-1, which seems to be important in adaptation strategies for coping with heat stress in this nematode.

Cytoplasmic HuR expression: correlation with cellular inhibitors of apoptosis protein-2 expression and clinicopathologic factors in oral squamous cell carcinoma cells.

BACKGROUND: HuR expression has been noted in several cancer types, in which it may contribute to increased expression of cellular inhibitors of apoptosis protein-2 (cIAP2) observed during tumorigenesis. METHODS: To assess the correlation between cIAP2 and HuR in cases of oral squamous cell carcinoma (OSCC), the expression patterns of HuR and cIAP2 were assessed by immunohistochemical analyses of 95 treated OSCC samples. RESULTS: In the tumor tissues, positive cytoplasmic HuR expression was evident in 71.6% of samples and positive cIAP2 expression was noted in 95.8% of samples. Positive cytoplasmic HuR expression was significantly associated with positive cIAP2 (p < .035) and high cIAP2 expression (p < .007), as well as high grade (p < .050). The inhibition of HuR expression by small interfering RNA or leptomycin B caused a reduction in the inducibility of cIAP2 in oral cancer cells. CONCLUSION: Cytoplasmic expression of HuR is associated with cIAP2 expression in OSCCs.

Development of a method to quantitate nematode pheromone for study of small-molecule metabolism in Caenorhabditis elegans.

Pheromones produced by Caenorhabditis elegans are considered key regulators of development, mating, and social behaviors in this organism. Here, we present a rapid mass spectrometry-based method (PheroQu) for absolute quantitation of nematode pheromones (e.g., daumone 1, 2, and 3) both in C. elegans worm bodies (as few as 20 worms) and in liquid culture medium. Pheromones were separated by ultra performance liquid chromatography and monitored by a positive electrospray ionization detector in the multiple-reaction monitoring mode. The daf-22 mutant worms were used as surrogate matrix for calibration, and stable deuterated isotope-containing pheromone was used as internal standard for measuring changes in pheromones in N2 wild-type and other strains under different growth conditions. The worm-body pheromones were extracted by acidified acetonitrile solvent, and the secreted pheromones were extracted from culture medium with solid-phase extraction cartridges. The run time was achieved in less than 2 min. The method was validated for specificity, linearity, accuracy, precision, recovery, and stability. The assay was linear over an amount range of 2-250 fmol, and the limit of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both worm bodies and culture medium. With the PheroQu method, we were able to identify the location of pheromone biosynthesis and determine the changes in different pheromone types synthesized, according to developmental stages and aging process. This method, which is simple, rapid, sensitive, and specific, will be useful for the study of small-molecule metabolism during developmental stages of C. elegans.

Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons.

Many nematodes show a stage-specific behavior called nictation in which a worm stands on its tail and waves its head in three dimensions. Here we show that nictation is a dispersal behavior regulated by a specific set of neurons, the IL2 cells, in C. elegans. We established assays for nictation and showed that cholinergic transmission was required for nictation. Cell type-specific rescue experiments and genetic ablation experiments revealed that the IL2 ciliated head neurons were essential for nictation. Intact cilia in IL2 neurons, but not in other ciliated head neurons, were essential, as the restoration of the corresponding wild-type gene activity in IL2 neurons alone in cilia-defective mutants was sufficient to restore nictation. Optogenetic activation of IL2 neurons induced nictation, suggesting that signals from IL2 neurons are sufficient for nictation. Finally, we demonstrated that nictation is required for transmission of C. elegans to a new niche using flies as artificial carriers, suggesting a role of nictation as a dispersal and survival strategy under harsh conditions.

Methods for evaluating the Caenorhabditis elegans dauer state: standard dauer-formation assay using synthetic daumones and proteomic analysis of O-GlcNAc modifications.

The dauer state is a non-feeding, alternative L3 state characterized by a number of distinctive metabolic and morphological changes. There are many naturally occurring dauer-inducing pheromones, termed daumones, that have been suggested by some to exhibit differences in dauer-inducing activity. Here, we have established a standard dauer-formation assay that uses synthetic daumones 1, 2, and 3, the three major daumones. To analyze the proteome of Caenorhabditis elegans in the dauer state, we focused on O-GlcNAc modification, a cytosolic modification of proteins that is known to interact either competitively or synergistically with protein phosphorylation. Protein O-GlcNAc modification is an important biological process in cells that can ensure the timely response to extracellular stimuli, such as daumone, and maintain cellular homeostasis. Establishing a standard method for assaying dauer formation using different synthetic daumones, and using differences in O-GlcNAcylated proteins during the dauer state to analyze the dauer proteome will lead to a better understanding of dauer biology of C. elegans in the context of animal longevity and adaptation under harsh environments.

STR-33, a novel G protein-coupled receptor that regulates locomotion and egg laying in Caenorhabditis elegans.

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gα(q) signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gα(o) signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gα(o). Endogenous acetylcholine suppressed egg laying in C. elegans via a Gα(o)-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

A potential biochemical mechanism underlying the influence of sterol deprivation stress on Caenorhabditis elegans longevity.

To investigate the biochemical mechanism underlying the effect of sterol deprivation on longevity in Caenorhabditis elegans, we treated parent worms (P0) with 25-azacoprostane (Aza), which inhibits sitosterol-to-cholesterol conversion, and measured mean lifespan (MLS) in F2 worms. At 25 µM (∼EC(50)), Aza reduced total body sterol by 82.5%, confirming sterol depletion. Aza (25 µM) treatment of wild-type (N2) C. elegans grown in sitosterol (5 µg/ml) reduced MLS by 35%. Similar results were obtained for the stress-related mutants daf-16(mu86) and gas-1(fc21). Unexpectedly, Aza had essentially no effect on MLS in the stress-resistant daf-2(e1370) or mitochondrial complex II mutant mev-1(kn1) strains, indicating that Aza may target both insulin/IGF-1 signaling (IIS) and mitochondrial complex II. Aza increased reactive oxygen species (ROS) levels 2.7-fold in N2 worms, but did not affect ROS production by mev-1(kn1), suggesting a direct link between Aza treatment and mitochondrial ROS production. Moreover, expression of the stress-response transcription factor SKN-1 was decreased in amphid neurons by Aza and that of DAF-28 was increased when DAF-6 was involved, contributing to lifespan reduction.

Contribution of the peroxisomal acox gene to the dynamic balance of daumone production in Caenorhabditis elegans.

Dauer pheromones or daumones, which are signaling molecules that interrupt development and reproduction (dauer larvae) during unfavorable growth conditions, are essential for cellular homeostasis in Caenorhabditis elegans. According to earlier studies, dauer larva formation in strain N2 is enhanced by a temperature increase, suggesting the involvement of a temperature-dependent component in dauer pheromone biosynthesis or sensing. Several naturally occurring daumone analogs (e.g. daumones 1-3) have been identified, and these molecules are predicted to be synthesized in different physiological settings in this nematode. To elucidate the molecular regulatory system that may influence the dynamic balance of specific daumone production in response to sudden temperature changes, we characterized the peroxisomal acox gene encoding acyl-CoA oxidase, which is predicted to catalyze the first reaction during biosynthesis of the fatty acid component of daumones. Using acox-1(ok2257) mutants and a new, robust analytical method, we quantified the three most abundant daumones in worm bodies and showed that acox likely contributes to the dynamic production of various quantities of three different daumones in response to temperature increase, changes that are critical in C. elegans for coping with the natural environmental changes it faces.

Caenorhabditis elegans utilizes dauer pheromone biosynthesis to dispose of toxic peroxisomal fatty acids for cellular homoeostasis.

Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.

Design, synthesis, and discovery of stilbene derivatives based on lithospermic acid B as potent protein tyrosine phosphatase 1B inhibitors.

Dihydroxy stilbene derivatives were designed based on lithospermic acid B and were prepared from 4-(chloromethyl)benzoic acid. The inhibitory activities of the novel compounds against protein tyrosine phosphatase 1B (PTP1B) were evaluated. 3,4-Dihydroxy stilbene carbonyl compounds (7, 11b, 27b) inhibited PTP1B with IC50 values comparable to molybdate, while the conjugation-extended compound (15b) showed inhibition 3-fold better than preclinical RK682. The introduction of electron withdrawing groups or amides into the second phenyl ring, or extension of the conjugation into the stilbene molecule may increase stability of the generated radicals.

Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone.

Pheromones are cell type-specific signals used for communication between individuals of the same species. When faced with overcrowding or starvation, Caenorhabditis elegans secrete the pheromone daumone, which facilitates communication between individuals for adaptation to adverse environmental stimuli. Daumone signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended life. Because daumone is a key regulator of chemosensory processes in development and ageing, the chemical identification of daumone is important for elucidating features of the daumone-mediated signalling pathway. Here we report the isolation of natural daumone from C. elegans by large-scale purification, as well as the total chemical synthesis of daumone. We present the stereospecific chemical structure of purified daumone, a fatty acid derivative. We demonstrate that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. These results should help to elucidate the daumone-mediated signalling pathway, which might in turn influence ageing and obesity research and the development of antinematodal drugs.