Color-encoded multicompartmental hydrogel microspheres for multiplexed bioassays.

We develop color-encoded multicompartmental hydrogel (MH) microspheres tailored for multiplexed bioassays using a drop-based microfluidic approach. Our method involves the creation of triple emulsion drops that feature thin sacrificial oil layers separating two prepolymer phases. This configuration leads to the formation of poly(ethylene glycol) (PEG) multi-compartmental core-shell microspheres through photopolymerization, followed by the removal of the thin oil layers. The core compartments stably incorporate pigments, ensuring their retention within the hydrogel network without leakage, which facilitates reliable color encoding across varying spatial positions. Additionally, we introduce small molecule fluorescent labeling into the chemically functionalized shell compartments, achieving consistent distribution of functional components without the core's contamination. Importantly, our integrated one-pot conjugation of these color-encoded microspheres with affinity peptides enables the highly sensitive and selective detection of influenza virus antigens using a fluorescence bioassay, resulting in an especially low detection limit of 0.18 nM and 0.66 nM for influenza virus H1N1 and H5N1 antigens, respectively. This approach not only highlights the potential of our microspheres in clinical diagnostics but also paves the way for their application in a wide range of multiplexed assays.

Mimicking chronic alcohol effects through a controlled and sustained ethanol release device.

Alcohol consumption, a pervasive societal issue, poses considerable health risks and socioeconomic consequences. Alcohol-induced hepatic disorders, such as fatty liver disease, alcoholic hepatitis, chronic hepatitis, liver fibrosis, and cirrhosis, underscore the need for comprehensive research. Existing challenges in mimicking chronic alcohol exposure in cellular systems, attributed to ethanol evaporation, necessitate innovative approaches. In this study, we developed a simple, reusable, and controllable device for examining the physiological reactions of hepatocytes to long-term alcohol exposure. Our approach involved a novel device designed to continuously release ethanol into the culture medium, maintaining a consistent ethanol concentration over several days. We evaluated device performance by examining gene expression patterns and cytokine secretion alterations during long-term exposure to ethanol. These patterns were correlated with those observed in patients with alcoholic hepatitis. Our results suggest that our ethanol-releasing device can be used as a valuable tool to study the mechanisms of chronic alcohol-mediated hepatic diseases at the cellular level. Our device offers a practical solution for studying chronic alcohol exposure, providing a reliable platform for cellular research. This innovative tool holds promise for advancing our understanding of the molecular processes involved in chronic alcohol-mediated hepatic diseases. Future research avenues should explore broader applications and potential implications for predicting and treating alcohol-related illnesses.

Solvent-Free Fabrication of Anisotropic Microparticles with Precise 3D Shape Control Using Dipping-Based Micromolding.

We present an innovative solvent-free micromolding technique for rapidly fabricating complex polymer microparticles with three-dimensional (3D) shapes utilizing a surface tension-induced dipping process. Our fabrication process involves loading a photocurable solution into micromolds through mold dipping. The loaded solution, induced by surface tension, undergoes spatial deformation upon mold removal caused by surface forces, ultimately acquiring an anisotropic shape before photopolymerization. Results show that the amount of photocurable solution loaded depends on the degree of capillary penetration, which can be adjusted by varying the dipping time and mold height. It enables the production of polymer particles with precisely controlled 3D shapes without diluting them with volatile organic solvents. Sequential micromolding enables the spatial stacking of the polymer domain through a bottom-up approach, facilitating the creation of complex multicompartmental microparticles with independently controlled compartments. Finally, we demonstrated the successful simultaneous conjugation of multiple model-fluorescent proteins through the biofunctionalization of microparticles, indicating functional stability and effective conjugation of hydrophilic molecules such as proteins. We also extend our capacity to create bicompartmental microparticles with distinct functionalities in each compartment, revealing spatially controlled functional structures. In summary, these findings demonstrate a straightforward, rapid, and reliable method for producing highly uniform complex particles with precise control over the 3D shape and compartmentalization, all accomplished without the use of organic solvents.

Electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta.

This paper reports the development of a highly sensitive and selective electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta (IL-1ß). To this end, flower-like Au-Ag@MoS2-rGO nanocomposites were used as the signal amplification platform to achieve a label-free biosensor with a high sensitivity and selectivity. First, a high-affinity peptide for IL-1ß was identified through biopanning with M13 random peptide libraries, and was newly designed by incorporating cysteine at the C-terminus. An IL-1ß specific binding peptide was used as the bio-receptor, and the interaction between the IL-1ß binding peptide and IL-1ß was confirmed via enzyme-linked immunosorbent assay and various physicochemical and electrochemical analyses. Under optimal conditions, the biosensor achieved an ultrasensitive and specific IL-1ß detection in a wide linear concentration range of 0-250 ng/mL with a picomolar-level detection limit (∼2.4 pM), low binding constant (∼0.62 pM), and a low coefficient of variation (<1.65 %). The biosensor was successfully utilized for IL-1ß determination in the serum of Crohn's disease patients with a good correlation coefficient. In addition, the detection performance was comparable to that of commercially available IL-1ß ELISA kit. This indicates that the electrochemical peptide-based biosensor may offer a potentially valuable platform for the clinical diagnosis of various inflammatory disease biomarkers.

Sulfur-doped graphitic carbon nitride: Tailored nanostructures for photocatalytic, sensing, and energy storage applications.

Rapid globalization and industrialization have led to widespread pollution and energy crises, necessitating the development of innovative solutions. Metal-free g-C3N4-based polymeric materials have unique properties but face limitations such as low surface area and inefficient light absorption. Doping, especially sulfur doping, is a prevalent technique to enhance their optical and electronic properties. This comprehensive review focuses on the synthesis techniques employed for sulfur doping of g-C3N4 (S-CN), highlighting the complexities associated with S-doping and the advantages of co-doping. Additionally, the review encompasses the diverse applications of S-CN in catalysis, photocatalysis, sonocatalysis, pollutant remediation, and electrochemical sensing. By incorporating sulfur into the g-C3N4 structure, various desirable properties can be achieved, including improved light absorption efficiency and enhanced charge carrier separation and migration. These advancements have broadened the application potential of S-CN in a range of important fields. S-CN has shown promise as a catalyst, facilitating various chemical reactions, as well as a photocatalyst, harnessing solar energy for environmental remediation and energy conversion processes. Moreover, S-CN exhibits potential in sonocatalysis for ultrasound-mediated reactions, pollutant remediation, and electrochemical sensing applications.

Affinity Peptide-Tethered Suspension Hydrogel Sensor for Selective and Sensitive Detection of Influenza Virus.

Influenza viruses are known to cause pandemic flu outbreaks through both inter-human and animal-to-human transmissions. Therefore, the rapid and accurate detection of such pathogenic viruses is crucial for effective pandemic control. Here, we introduce a novel sensor based on affinity peptide-immobilized hydrogel microspheres for the selective detection of influenza A virus (IAV) H3N2. To enhance the binding affinity performance, we identified novel affinity peptides using phage display and further optimized their design. The functional hydrogel microspheres were constructed using the drop microfluidic technique, employing a structure composed of natural (chitosan) and synthetic (poly(ethylene glycol) diacrylate and PEG 6 kDa) polymers with the activation of azadibenzocyclooctyne for the subsequent click chemistry reaction. The binding peptide-immobilized hydrogel microsphere (BP-Hyd) was characterized by field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy and exhibited selective detection capability for the IAV H3N2. To capture the detected IAV H3N2, a Cy3-labeled IAV hemagglutinin antibody was utilized. By incorporating the affinity peptide with hydrogel microspheres, we achieved quantitative and selective detection of IAV H3N2 with a detection limit of 1.887 PFU mL-1. Furthermore, the developed suspension sensor exhibited excellent reproducibility and showed reusability potential. Our results revealed that the BP-Hyd-based fluorescence sensor platform could be feasibly employed to detect other pathogens because the virus-binding peptides can be easily replaced with other peptides through phage display, enabling selective and sensitive binding to different targets.

Broad-temperature-range mechanically tunable hydrogel microcapsules for controlled active release.

Here, we report PNIPAm-co-PEGDA hydrogel shelled microcapsules with a thin oil layer to achieve tunable thermo-responsive release of the encapsulated small hydrophilic actives. We use a microfluidic device integrated with a temperature-controlled chamber for consistent and reliable production of the microcapsules by utilizing triple emulsion drops (W/O/W/O) with a thin oil layer as capsule templates. The interstitial oil layer between the aqueous core and the PNIPAm-co-PEGDA shell provides a diffusion barrier for the encapsulated active until the temperature reaches a critical point above which the destabilization of interstitial oil layer occurs. We find that the destabilization of the oil layer with temperature increase is caused by outward expansion of the aqueous core due to volume increase and the radial inward compression from the deswelling of the thermo-responsive hydrogel shell. The copolymerization of NIPAm with PEGDA increases the biocompatibility of the resulting microcapsule while offering the ability to alter the compressive modulus in broad ranges by simply varying crosslinker concentrations thereby to precisely tune the onset release temperature. Based on this concept, we further demonstrate that the release temperature can be enhanced up to 62 °C by adjusting the shell thickness even without varying the chemical composition of the hydrogel shell. Moreover, we incorporate gold nanorods within the hydrogel shell to spatiotemporally regulate the active release from the microcapsules by illuminating with non-invasive near infrared (NIR) light.

Harnessing protein sensing ability of electrochemical biosensors via a controlled peptide receptor-electrode interface.

BACKGROUND: Cathepsin B, a cysteine protease, is considered a potential biomarker for early diagnosis of cancer and inflammatory bowel diseases. Therefore, more feasible and effective diagnostic method may be beneficial for monitoring of cancer or related diseases. RESULTS: A phage-display library was biopanned against biotinylated cathepsin B to identify a high-affinity peptide with the sequence WDMWPSMDWKAE. The identified peptide-displaying phage clones and phage-free synthetic peptides were characterized using enzyme-linked immunosorbent assays (ELISAs) and electrochemical analyses (impedance spectroscopy, cyclic voltammetry, and square wave voltammetry). Feasibilities of phage-on-a-sensor, peptide-on-a-sensor, and peptide-on-a-AuNPs/MXene sensor were evaluated. The limit of detection and binding affinity values of the peptide-on-a-AuNPs/MXene sensor interface were two to four times lower than those of the two other sensors, indicating that the peptide-on-a-AuNPs/MXene sensor is more specific for cathepsin B (good recovery (86-102%) and %RSD (< 11%) with clinical samples, and can distinguish different stages of Crohn's disease. Furthermore, the concentration of cathepsin B measured by our sensor showed a good correlation with those estimated by the commercially available ELISA kit. CONCLUSION: In summary, screening and rational design of high-affinity peptides specific to cathepsin B for developing peptide-based electrochemical biosensors is reported for the first time. This study could promote the development of alternative antibody-free detection methods for clinical assays to test inflammatory bowel disease and other diseases.

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system.

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L-1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione /gglucose , respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.

Capsule-based colorimetric temperature monitoring system for customizable cold chain management.

The COVID-19 pandemic and the resulting supply chain disruption have rekindled crucial needs for safe storage and transportation of essential items. Despite recent advances, existing temperature monitoring technologies for cold chain management fall short in reliability, cost, and flexibility toward customized cold chain management for various products with different required temperature. In this work, we report a novel capsule-based colorimetric temperature monitoring system with precise and readily tunable temperature ranges. Triple emulsion drop-based microfluidic technique enables rapid production of monodisperse microcapsules with an interstitial phase-change oil (PCO) layer with precise control over its dimension and composition. Liquid-solid phase transition of the PCO layer below its freezing point triggers the release of the encapsulated payload yielding drastic change in color, allowing user-friendly visual monitoring in a highly sensitive manner. Simple tuning of the PCO layer's compositions can further broaden the temperature range in a precisely controlled manner. The proposed simple scheme can readily be formulated to detect both temperature rise in the frozen environment and freeze detection as well as multiple temperature monitoring. Combined, these results support a significant step forward for the development of customizable colorimetric monitoring of a broad range of temperatures with precision.

Grifola frondosa Extract Containing Bioactive Components Blocks Skin Fibroblastic Inflammation and Cytotoxicity Caused by Endocrine Disrupting Chemical, Bisphenol A.

Grifola frondosa (GF), a species of Basidiomycotina, is widely distributed across Asia and has been used as an immunomodulatory, anti-bacterial, and anti-cancer agent. In the present study, the pharmacological activity of the GF extract against an ecotoxicological industrial chemical, bisphenol A (BPA) in normal human dermal fibroblasts (NHDFs), was investigated. GF extract containing naringin, hesperidin, chlorogenic acid, and kaempferol showed an inhibitory effect on cell death and inflammation induced by BPA in the NHDFs. For the cell death caused by BPA, GF extract inhibited the production of reactive oxygen species responsible for the unique activation of the extracellular signal-regulated kinase. In addition, GF extract attenuated the expression of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and the pro-inflammatory cytokine IL-1ß by the suppression of the redox-sensitive transcription factor, nuclear factor-kappa B (NF-κB) in BPA-treated NHDFs. For the inflammation triggered by BPA, GF extract blocked the inflammasome-mediated caspase-1 activation that leads to the secretion of IL-1ß protein. These results indicate that the GF extract is a functional antioxidant that prevents skin fibroblastic pyroptosis induced by BPA.

Fine-tuning of MXene-nickel oxide-reduced graphene oxide nanocomposite bioelectrode: Sensor for the detection of influenza virus and viral protein.

Influenza viruses can cause epidemics through inter-human transmission, and the social consequences of viral transmission are incalculable. Current diagnostics for virus detection commonly relies on antibodies or nucleic acid as recognition reagent. However, a more advanced and general method for the facile development of new biosensors is increasing in demand. In this study, we report the fabrication of an ultra-sensitive peptide-based nanobiosensor using a nickel oxide (NiO)-reduced graphene oxide (rGO)/MXene nanocomposite to detect active influenza viruses (H1N1 and H5N2) and viral proteins. The sensing mechanism is based on the signal inhibition, the specific interaction between H1N1 (QMGFMTSPKHSV) and H5N1 (GHPHYNNPSLQL) binding peptides anchored on the NiO-rGO/MXene/glassy carbon electrode (GCE) surface and the viral surface protein hemagglutinin (HA) is the critical factor for the decrease in the peak current of the sensor. In this strategy, the NiO-rGO/MXene nanocomposite results in synergistic signal effects, including electrical conductivity, porosity, electroactive surface area, and active site availability when viruses are deposited on the electrode. Based on these observations, the results showed that the developed nanobiosensor was capable of highly sensitive and specific detection of their corresponding influenza viruses and viral proteins with a very low detection limit (3.63 nM of H1N1 and 2.39 nM for H5N1, respectively) and good recovery. The findings demonstrate that the proposed NiO-rGO/MXene-based peptide biosensor can provide insights for developing a wide range of clinical screening tools for detecting affected patients.

Dual synergistic response for the electrochemical detection of H1N1 virus and viral proteins using high affinity peptide receptors.

Identifying alternatives to antibodies as bioreceptors to test samples feasibly is crucial for developing next-generation in vitro diagnostic methods. Here, we aimed to devise an analytical method for detecting H1N1 viral proteins (hemagglutinin [HA] and neuraminidase [NA]) as well as the complete H1N1 virus with high sensitivity and selectivity. By applying biopanning of M13 peptide libraries, high affinity peptides specific for HA or NA were successfully identified. After selection, three different synthetic peptides that incorporated gold-binding motifs were designed and chemically synthesized on the basis of the original sequence identified phage display technique with or without two repeat. Their binding interactions were characterized by enzyme-linked immunosorbent assay (ELISA), square wave voltammetry (SWV), Time of flight-secondary ion mass spectroscopy (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). The binding constants (Kd) of HA BP1, HA BP2 and NA BP1 peptides were found to be 169.72 nM, 70.02 nM and 224.49 nM for HA or NA proteins by electrochemical measurements (SWV). The single use of HA BP2 peptide enabled the detection of either H1N1 viral proteins or the actual H1N1 virus, while NA BP1 peptide exhibited lower binding for real H1N1 virus particles. Moreover, the use of both HA BP1 and BP2 as a divalent capturing reagent improved sensor performance as well as the strength of the electrochemical signal, thereby exhibiting a dual synergistic effect for the electrochemical detection of H1N1 antigens with satisfactory specificity and sensitivity (limit of detection of 1.52 PFU/mL).

Oral delivery of stem-cell-loaded hydrogel microcapsules restores gut inflammation and microbiota.

Mesenchymal stem cells (MSCs) are an attractive candidate for the treatment of inflammatory bowel disease (IBD), but their poor delivery rate to an inflamed colon is a major factor hampering the clinical potential of stem cell therapies. Moreover, there remains a formidable hurdle to overcome with regard to survival and homing in to injured sites. Here, we develop a strategy utilizing monodisperse hydrogel microcapsules with a thin intermediate oil layer prepared by a triple-emulsion drop-based microfluidic approach as an in-situ oral delivering carrier. The oral delivery of stem-cell-loaded hydrogel microcapsules (SC-HM) enhances MSC survival and retention in the hostile stomach environment due to the intermediate oil layer and low value of the overall stiffness, facilitating programmable cell release during gastrointestinal peristalsis. SC-HM is shown to induce tissue repair, reduce the colonic macrophage infiltration responsible for the secretion of the pro-inflammatory factors, and significantly mitigate the severity of IBD in a mouse model, where MSCs released by SC-HM successfully accumulate at the colonic crypt. Moreover, a metagenomics analysis reveals that SC-HM ameliorates the dysbiosis of specific bacterial genera, including Bacteroides acidifaciens, Lactobacillus (L.) gasseri, Lactobacillus reuteri, and L. intestinalis, implying optimization of the microorganism's composition and abundance. These findings demonstrate that SC-HM is a potential IBD treatment candidate.

Strategies, advances, and challenges associated with the use of graphene-based nanocomposites for electrochemical biosensors.

Graphene is an intriguing two-dimensional honeycomb-like carbon material with a unique basal plane structure, charge carrier mobility, thermal conductivity, wide electrochemical spectrum, and unusual physicochemical properties. Therefore, it has attracted considerable scientific interest in the field of nanoscience and bionanotechnology. The high specific surface area of graphene allows it to support high biomolecule loading for good detection sensitivity. As such, graphene, graphene oxide (GO), and reduced GO are excellent materials for the fabrication of new nanocomposites and electrochemical sensors. Graphene has been widely used as a chemical building block and/or scaffold with various materials to create highly sensitive and selective electrochemical sensing microdevices. Over the past decade, significant advancements have been made by utilizing graphene and graphene-based nanocomposites to design electrochemical sensors with enhanced analytical performance. This review focus on the synthetic strategies, as well as the structure-to-function studies of graphene, electrochemistry, novel multi nanocomposites combining graphene, limit of detection, stability, sensitivity, assay time. Finally, the review describes the challenges, strategies and outlook on the future development of graphene sensors technology that would be usable for the internet of things are also highlighted.

Electrochemical detection of caspase-3 based on a chemically modified M13 phage virus.

Caspase-3, a cysteine-dependent protease, is considered a reliable molecular biomarker for the diagnosis and prognosis of apoptosis-related diseases. In this study, we demonstrated a phage-based electrochemical biosensor for the evaluation of cell apoptosis by the sensitive and specific detection of caspase-3. Specifically, for screening of affinity peptide-displayed phages, phage display was performed using M13 phage libraries (cyclic forms of peptides), and we identified potential affinity peptide-displayed phage clones with the sequence CPTTMWRYC. After characterization of its binding affinity using enzyme-linked immunosorbent assay, whole phage particles were covalently attached to a gold surface using coupling chemistry (MUA-EDC/NHS). The developed phage sensor was characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscopy (SEM), electrochemical analysis using cyclic voltammetry (CV), and square wave voltammetry (SWV). Under optimal conditions, the affinity peptide-displayed phage sensor showed a good binding affinity (Kd = 0.13 ± 0.56 µM) and limit of detection (0.39 µM) for caspase-3 detection. Furthermore, developed phage sensor could be monitored the response of apoptotic HeLa cells by detecting caspase-3 activity. This work should stimulate the development of efficient alternative caspase-3 detection methods for the diagnosis and prognosis of apoptosis-related diseases.

Cell-Inspired Hydrogel Microcapsules with a Thin Oil Layer for Enhanced Retention of Highly Reactive Antioxidants.

In nature, individual cells are compartmentalized by a membrane that protects the cellular elements from the surrounding environment while simultaneously equipped with an antioxidant defense system to alleviate the oxidative stress resulting from light, oxygen, moisture, and temperature. However, this mechanism has not been realized in cellular mimics to effectively encapsulate and retain highly reactive antioxidants. Here, we report cell-inspired hydrogel microcapsules with an interstitial oil layer prepared by utilizing triple emulsion drops as templates to achieve enhanced retention of antioxidants. We employ ionic gelation for the hydrogel shell to prevent exposure of the encapsulated antioxidants to free radicals typically generated during photopolymerization. The interstitial oil layer in the microcapsule serves as an stimulus-responsive diffusion barrier, enabling efficient encapsulation and retention of antioxidants by providing an adequate pH microenvironment until osmotic pressure is applied to release the cargo on-demand. Moreover, addition of a lipophilic reducing agent in the oil layer induces a complementary reaction with the antioxidant, similar to the nonenzymatic antioxidant defense system in cells, leading to enhanced retention of the antioxidant activity. Furthermore, we show the complete recovery and even further enhancement in antioxidant activity by lowering the storage temperature, which decreases the oxidation rate while retaining the complementary reaction with the lipophilic reducing agent.

Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency.

BACKGROUND AND AIM: Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway. MAIN METHODS AND MAJOR RESULTS: The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLA2 strain expressing phospholipase A2 (PLA2 ) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P-PLA2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di-FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR-YA/FF+P-PLA2 strain resulted in the production of 1.22 ± 0.01 g L-1 Di-FL and 0.47 ± 0.01 g L-1 2'-FL, whereas the control strain could only produce 0.65 ± 0.01 g L-1 2'-FL. CONCLUSIONS AND IMPLICATIONS: This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

Kaempferol Blocks the Skin Fibroblastic Interleukin 1ß Expression and Cytotoxicity Induced by 12-O-tetradecanoylphorbol-13-acetate by Suppressing c-Jun N-terminal Kinase.

Kaempferol, a bioflavonoid present in fruits and vegetables, has a variety of antioxidant and anti-inflammatory capacities, but the functional role of kaempferol in oxidative skin dermal damage has yet to be well studied. In this study, we examine the role of kaempferol during the inflammation and cell death caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human dermal fibroblasts (NHDF). TPA (5 µM) significantly induced cytotoxicity of NHDF, where a robust increase in the interleukin (IL)-1ß mRNA among the various pro-inflammatory cytokines. The skin fibroblastic cytotoxicity and IL-1ß expression induced by TPA were significantly ameliorated by a treatment with 100 nM of kaempferol. Kaempferol blocked the production of the intracellular reactive oxygen species (ROS) responsible for the phosphorylation of c-Jun N-terminal kinase (JNK) induced by TPA. Interestingly, we found that kaempferol inhibited the phosphorylation of nuclear factor-kappa B (NF-κB) and the inhibitor NF-κB (IκBα), which are necessary for the expression of cleaved caspase-3 and the IL-1ß secretion in TPA-treated NHDF. These results suggest that kaempferol is a functional agent that blocks the signaling cascade of the skin fibroblastic inflammatory response and cytotoxicity triggered by TPA.

Astaxanthin Inhibits Autophagic Cell Death Induced by Bisphenol A in Human Dermal Fibroblasts.

Astaxanthin, a natural antioxidant carotenoid, is a nutrient with diverse health benefits, given that it decreases the risk of oxidative stress-related diseases. In the present study, we investigate the functional role of astaxanthin during autophagic cell death induced by the estrogenic endocrine-disrupting chemical bisphenol A (BPA) in normal human dermal fibroblasts (NHDF). BPA significantly induced apoptotic cell death and autophagy in NHDF. Autophagic cell death evoked by BPA was significantly restored upon a treatment with astaxanthin (10 µM) via the inhibition of intracellular reactive oxygen species (ROS) production. Astaxanthin inhibited the phosphorylation of extracellular signal-regulated kinases (ERK) stimulated by ROS production, but it did not influence the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in BPA-treated NHDF. Astaxanthin abrogated the ERK-mediated activation of nuclear factor-kappa B (NF-κB), which is responsible for the mRNA expression of LC3-II, Beclin-1, Atg12, and Atg14 during apoptotic cell death induced by BPA. These results indicate that astaxanthin is a pharmacological and nutritional agent that blocks the skin fibroblastic autophagic cell death induced by BPA in human dermal fibroblasts.