Hormone-free vs. follicle-stimulating hormone-primed infertility treatment of women with polycystic ovary syndrome using biphasic in vitro maturation: a randomized controlled trial.

OBJECTIVE: To compare oocyte maturation rates and pregnancy outcomes in women with polycystic ovary syndrome (PCOS) undergoing biphasic in vitro maturation (capacitation in vitro maturation [CAPA-IVM]) with vs. without follicle-stimulating hormone (FSH) priming. DESIGN: Randomized, controlled, assessor-blinded trial. SETTING: Private hospital. PATIENT(S): Women aged 18-37 years with PCOS and an indication for CAPA-IVM. INTERVENTION(S): Participants were randomized (1:1) to undergo CAPA-IVM with or without FSH priming. The FSH priming group had 2 days of FSH injections before oocyte pickup; no FSH was given in the non-FSH group. After CAPA-IVM, day-5 embryos were vitrified for transfer in a subsequent cycle. MAIN OUTCOME MEASURE(S): The primary endpoint was number of matured oocytes. Secondary outcomes included rates of live birth, implantation, clinical pregnancy, ongoing pregnancy, pregnancy complications, obstetric and perinatal complications, and neonatal complications. RESULT(S): The number (interquartile range) of matured oocytes did not differ significantly in the non-FSH vs. FSH group (13 [9-18] vs. 14 [7-18]; absolute difference -1 [95% confidence interval -5 to 4]); other oocyte and embryology outcomes did not differ between groups. Rates of ongoing pregnancy and live birth were 38.3% in the non-FSH group and 31.7% in the FSH group (risk ratio for both outcomes: 1.21, 95% confidence interval 0.74-1.98). Maternal complications were infrequent and occurred at a similar rate in the two groups; there were no preterm deliveries before 32 weeks gestation. CONCLUSION(S): These findings open the possibility of a new, hormone-free approach to infertility treatment of women with PCOS.

Whole genome sequence analysis of Bacillus amyloliquefaciens strain S2.5 as a potential probiotic for feed supplement in livestock production.

BACKGROUND: Supplementing probiotics in livestock feed is increasing due to concerns over the potential harm caused by antibiotics and other chemical growth promoters. Several Bacillus sp. have been used as probiotic supplements for livestock. In this study, Bacillus amyloliquefaciens S2.5 was isolated from freshwater and its potential probiotic characteristics were evaluated in vitro. The whole genome of strain S2.5 was sequenced, and its probiotic traits were annotated using bioinformatic tools. RESULTS: Both vegetative cells and spores of strain S2.5 remained stable throughout the 1.5 h of gastric juice and 48 h of intestine simulation. The strain S2.5 harbored the ability to produce glucoamylase, carboxymethyl cellulase, protease, and chitinase. It is also susceptible to all six tested antibiotics. The complete genome sequence shows genes related to acid-bile tolerance, environmental stress resistance, hydrolases, and adhesion to gut mucosa, confirming probiotic traits in the in vitro experiments. CONCLUSIONS: B. amyloliquefaciens S2.5 demonstrated potential probiotic characteristics and its genetic profile in the in vitro experiments. Further in vivo assessments of B. amyloliquefaciens S2.5 on livestock and poultry should be performed to assess its practical application.

Paper/GO/e-Au flexible SERS sensors for in situ detection of tricyclazole in orange juice and on cucumber skin at the sub-ppb level: machine learning-assisted data analysis.

Despite being an excellent surface enhanced Raman scattering (SERS) active material, gold nanoparticles were difficult to be loaded onto the surface of filter paper to fabricate flexible SERS substrates. In this study, electrochemically synthesized gold nanoparticles (e-AuNPs) were deposited on graphene oxide (GO) nanosheets in solution by ultrasonication, resulting in the formation of a GO/Au hybrid material. Thanks to the support of GO, the hybrid material could adhere onto the surface of filter paper, which was immersed into a GO/Au solution for 24 h and dried naturally at room temperature. The paper-based materials were then employed as substrates for a surface enhanced Raman scattering (SERS) sensing platform to detect tricyclazole (TCZ), a widely used pesticide, resulting in better sensitivity compared to the use of paper/Au SERS sensors. With the most optimal GO content of 4%, paper/GO/Au SERS sensors could achieve a limit of detection of 1.32 × 10-10 M in standard solutions. Furthermore, the filter paper-based SERS sensors also exhibited significant advantages in sample collection in real samples. On one hand, the sensors were dipped into orange juice, allowing TCZ molecules in this real sample to be adsorbed onto their SERS active surface. On the other hand, they were pasted onto cucumber skin to collect the analytes. As a result, the paper/GO/Au SERS sensors could sense TCZ in orange juice and on cucumber skin at concentrations as low as 10-9 M (∼2 ppb). In addition, a machine learning model was designed and developed, allowing the sensing system to discriminate TCZ from nine other organic compounds and predict the presence of TCZ on cucumber skin at concentrations down to 10-9 M.

Surface ligand modified silver nanoparticles-based SERS sensing platform for ultrasensitive detection of the pesticide thiram in green tea leaves: roles of coating agents in sensing performance.

Silver nanoparticles (AgNPs) have been regarded as a highly promising substrate for surface-enhanced Raman scattering (SERS) sensors. In this study, we focused on the electrochemical synthesis method by developing three kinds of AgNPs using three different electrolytes: citrate (e-Ag-C), oleic acid (e-Ag-O) and fish mint (Houttuynia cordata Thunb.) extract (e-Ag-bio). The as-prepared AgNPs were characterized and then employed as SERS substrates to detect the pesticide thiram. The obtained results show that e-Ag-O exhibits the best SERS performance. The effect of the coating agent was explained by chemical and electromagnetic enhancements (CM and EM). Although thiram could absorb onto e-Ag-C at the highest level, allowing its Raman signal to be best enhanced via the CM, the smallest interparticle distance of e-Ag-O could have resulted in the largest improvement in the EM. Using e-Ag-O to develop SERS-based sensors for thiram, we obtain the impressive detection limit of 1.04 × 10-10 M in standard samples and 10-9 M in tea leaves. The linear ranges are from 10-4 M to 10-7 M and from 10-7 M to 10-9 M, covering the maximum residue levels for plant commodities established by the United States Environment Protection Agency and European Food Safety Authority (2-13 ppm ∼7.7 × 10-6 M to 5 × 10-5 M).

Overpromoted and underregulated: National binding legal measures related to commercially produced complementary foods in seven Southeast Asian countries are not fully aligned with available guidance.

The market for commercially produced complementary foods (CPCF) is rapidly expanding in Southeast Asia; however, the existence and content of mandatory national policies, standards and legislation (binding legal measures) for CPCF in the region is unclear. To assess the status of national binding legal measures for CPCF in Southeast Asia, a legal and policy desk review was conducted in seven countries (Cambodia, Laos People's Democratic Republic, Indonesia, Malaysia, Philippines, Thailand and Viet Nam). The alignment of the national binding legal measures relevant to CPCF was assessed against guidance on CPCF nutrient composition and labelling requirements provided by Codex Alimentarius and the World Health Organization (WHO). Each of the seven countries had at least two national binding legal measures related to the nutrient composition or labelling of CPCF; however, there was limited alignment with the guidance from Codex and WHO. No country was fully aligned with the three CPCF-specific Codex standards/guidelines and only one country was in full alignment with the recommendations related to the protection of breastfeeding from the 'WHO Guidance on ending the inappropriate promotion of foods for infants and young children'. The findings of the review indicate that the existing national binding legal measures are insufficient to ensure that the CPCF sold as suitable for older infants and young children are nutritionally adequate and labelled in a responsible manner that does not mislead caregivers. Improved and enforced national binding legal measures for CPCF, in alignment with global guidance, are required to ensure that countries protect, promote and support optimal nutrition for children 6-36 months of age.

Enhanced sensing performance of carbaryl pesticide by employing a MnO2/GO/e-Ag-based nanoplatform: role of graphene oxide as an adsorbing agent in the SERS analytical performance.

A functional ternary substrate was developed for surface-enhanced Raman scattering (SERS) sensing systems. MnO2 nanosheets were synthesized by a simple and controllable hydrothermal method, followed by the integration of graphene oxide (GO) nanosheets. Subsequently, MnO2/GO nanostructures were decorated with plasmonic Ag nanoparticles (e-AgNPs). The MnO2/GO/e-Ag substrate could enhance the SERS sensing signal for organic chemicals without the assistance of chemical bonds between those analytes and the semiconductor within the ternary substrate, which have been proven to promote charge transfer and elevate the SERS enhancement in previous studies. Instead, GO nanosheets acted as a carpet also supporting the MnO2 nanosheets and e-AgNPs to form a porous structure, allowing the analytes to be well-adsorbed onto the ternary substrate, which improved the sensing performance of the SERS platform, compared to pure e-AgNPs, MnO2/e-Ag, and GO/e-Ag alone. The GO content in the nanocomposite was also considered to optimize the SERS substrate. With the most optimal GO content of 0.1 wt%, MnO2/GO/e-Ag-based SERS sensors could detect carbaryl, a pesticide, at concentrations as low as 1.11 × 10-8 M in standard solutions and 10-7 M in real tap water and cucumber extract.

Automated classification of giant virus genomes using a random forest model built on trademark protein families.

Viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are prevalent in various environments around the globe and play significant roles in shaping eukaryotic diversity and activities in global ecosystems. Given the extensive phylogenetic diversity within this viral group and the highly complex composition of their genomes, taxonomic classification of giant viruses, particularly incomplete metagenome-assembled genomes (MAGs) can present a considerable challenge. Here we developed TIGTOG (Taxonomic Information of Giant viruses using Trademark Orthologous Groups), a machine learning-based approach to predict the taxonomic classification of novel giant virus MAGs based on profiles of protein family content. We applied a random forest algorithm to a training set of 1,531 quality-checked, phylogenetically diverse Nucleocytoviricota genomes using pre-selected sets of giant virus orthologous groups (GVOGs). The classification models were predictive of viral taxonomic assignments with a cross-validation accuracy of 99.6% to the order level and 97.3% to the family level. We found that no individual GVOGs or genome features significantly influenced the algorithm's performance or the models' predictions, indicating that classification predictions were based on a comprehensive genomic signature, which reduced the necessity of a fixed set of marker genes for taxonomic assigning purposes. Our classification models were validated with an independent test set of 823 giant virus genomes with varied genomic completeness and taxonomy and demonstrated an accuracy of 98.6% and 95.9% to the order and family level, respectively. Our results indicate that protein family profiles can be used to accurately classify large DNA viruses at different taxonomic levels and provide a fast and accurate method for the classification of giant viruses. This approach could easily be adapted to other viral groups.

Ultrasensitive detection of crystal violet using a molybdenum sulfide-silver nanostructure-based sensing platform: roles of the adsorbing semiconductor in SERS signal enhancement.

Crystal violet (CV) is an organic dye that is stabilized by the extensive resonance delocalization of electrons over three electron-donating amine groups. This prevents the molecule from being linked to a metal surface, and therefore, reduces the sensitivity of surface-enhanced Raman scattering (SERS) sensors for this toxic dye. In this work, we improved the sensing performance of a silver-based SERS sensor for CV detection by modifying the active substrate. Molybdenum sulfide (MoS2) nanosheets were employed as a scaffold for anchoring electrochemically synthesized silver nanoparticles (e-AgNPs) through a single step of ultrasonication, leading to the formation of MoS2/Ag nanocomposites. As an excellent adsorbent, MoS2 promoted the adsorption of CV onto the surface of the substrate, allowing more CV molecules to be able to experience the SERS effect originating from the e-AgNPs. Hence, the SERS signal of CV was significantly enhanced. In addition, the effects of the MoS2 content of the nanocomposites on their SERS performance were also taken into account. Using MoS2/Ag with the most optimal MoS2 content of 10%, the SERS sensor exhibited the best enhancement of the SERS signal of CV with an impressive detection limit of 1.17 × 10-11 M in standard water and 10-9 M in tap water thanks to an enhancement factor of 2.9 × 106, which was 11.2 times higher than that using pure e-AgNPs.

The International Virus Bioinformatics Meeting 2023.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

Gold and Iron Oxide Nanoparticle Assemblies on Turnip Yellow Mosaic Virus for In-Solution Photothermal Experiments.

The ability to construct three-dimensional architectures via nanoscale engineering is important for emerging applications in sensors, catalysis, controlled drug delivery, microelectronics, and medical diagnostics nanotechnologies. Because of their well-defined and highly organized symmetric structures, viral plant capsids provide a 3D scaffold for the precise placement of functional inorganic particles yielding advanced hierarchical hybrid nanomaterials. In this study, we used turnip yellow mosaic virus (TYMV), grafting gold nanoparticles (AuNP) or iron oxide nanoparticles (IONP) onto its outer surface. It is the first time that such an assembly was obtained with IONP. After purification, the resulting nano-biohybrids were characterized by different technics (dynamic light scattering, transmission electron microcopy, X-ray photoelectron spectroscopy…), showing the robustness of the architectures and their colloidal stability in water. In-solution photothermal experiments were then successfully conducted on TYMV-AuNP and TYMV-IONP, the related nano-biohybrids, evidencing a net enhancement of the heating capability of these systems compared to their free NP counterparts. These results suggest that these virus-based materials could be used as photothermal therapeutic agents.

Encapsulation of Triphasia trifolia extracts by pH and thermal dual-sensitive chitosan hydrogels for controlled release.

Recent studies have developed varied delivery systems incorporating natural compounds to improve the limitations of plant extracts for clinical use while enabling their controlled release at treatment sites. For the first time, ethanolic limeberry extract (Triphasia trifolia) has been successfully encapsulated in thermo-sensitive chitosan hydrogels by a facile in situ loading. The extract-incorporated chitosan hydrogels have a pH value of nearly 7.00, gelation temperatures in the range of 37-38 °C, and exhibit an open-cell porous structure, thus allowing them to absorb and retain 756 % of their mass in water. The in vitro extract release from the hydrogels is driven by both temperature and pH, resulting in more than 70 % of the initial extract being released within the first 24 h. Although the release half-life of hydrogels at pH 7.4 is longer, their release capacity is higher than that at pH 6.5. Upon a 2 °C increase in temperature, the time to release 50 % initial extract is sharply reduced by 20-40 %. The release kinetics from the hydrogels mathematically demonstrated that diffusion is a prominent driving force over chitosan relaxation. Consequently, the developed hydrogels encapsulating the limeberry extract show their heat and pH sensitivity in controlled release for treating chronic wounds.

Virologs, viral mimicry, and virocell metabolism: the expanding scale of cellular functions encoded in the complex genomes of giant viruses.

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses" have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host-referred to here as viral homologs or "virologs"-as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates" immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.

A manganese dioxide-silver nanostructure-based SERS nanoplatform for ultrasensitive tricyclazole detection in rice samples: effects of semiconductor morphology on charge transfer efficiency and SERS analytical performance.

Taking advantage of metal-semiconductor junctions, functional nanocomposites have been designed and developed as active substrates for surface-enhanced Raman scattering (SERS) sensing systems. In this work, we prepared three types of nanocomposites based on manganese oxide (MnO2) nanostructures and electrochemically synthesized silver nanoparticles (e-AgNPs), which differed according to the morphologies of MnO2. The SERS performance of MnO2 nanosheet/e-Ag (MnO2-s/e-Ag), MnO2 nanorod/e-Ag (MnO2-r/e-Ag), and MnO2 nanowire/e-Ag (MnO2-w/e-Ag) was then evaluated using tricyclazole (TCZ), a commonly used pesticide, as an analyte. Compared to the others, MnO2-s/e-Ag exhibited the most remarkable SERS enhancement. Thanks to its large surface area and ability to accept/donate the electrons of the semiconductor, MnO2-s acted as a bridge to improve the charge transfer efficiency from e-Ag to TCZ. In addition, the MnO2 content of the nanocomposites was also considered to optimize the SERS sensing performance. With the optimal MnO2 content of 25 wt%, MnO2-s/e-Ag could achieve the best SERS performance, allowing the detection of TCZ at concentrations down to 6 × 10-12 M in standard solutions and 10-11 M in real rice samples.

Break to Build: Isothermal Assembly of Nucleic Acid Nanoparticles (NANPs) via Enzymatic Degradation.

The intrinsic properties of RNA and DNA biopolymers emphasized by engineered nucleic acid nanoparticles (NANPs) offer accelerated development of next-generation therapies. The rational design of NANPs facilitates programmable architectures intended for regulated molecular and cellular interactions. The conventional bottom-up assembly of NANPs relies on the thermal annealing of individual strands. Here, we introduce a concept of nuclease-driven production of NANPs where selective digestion of functionally inert structures leads to isothermal self-assembly of liberated constituents. The working principles, morphological changes, assembly kinetics, and the retention of structural integrity for system components subjected to anhydrous processing and storage are assessed. We show that the assembly of precursors into a single structure improves stoichiometry and enhances the functionality of nuclease-driven products. Furthermore, the experiments with immune reporting cell lines show that the developed protocols retain the immunostimulatory functionality of tested NANPs. The presented approach enables exploitation of the advantages of conditionally produced NANPs and demonstrates that NANPs' stability, immunorecognition, and assembly can be regulated to allow for a more robust functional system.

Evaluation of Ibuprofen Prolonged Release of Biomedical PLA-PEG-PLA Hydrogel via Degradation Mechanism.

A micellar hydrogel has long been considered an intelligent hydrophobic drug delivery material. In this study, synthesized PLA1750-PEG1750-PLA1750 micellar hydrogel aims to encapsulate ibuprofen (IBU) in the core PLA hydrophobic of the micelle and prolong the drug release time by an injectable route. The structure and morphology of the PLA1750-PEG1750-PLA1750 copolymer hydrogel were demonstrated by 1H NMR and TEM data. The hydrogel also achieved a gel state at a high concentration of 25 wt.% under the physiological conditions of the body (37°C, pH 7.4). Besides, the biocompatibility test displayed that the hydrogel slightly affected mice after injection one week and fully recovered after four weeks. Furthermore, the in vitro degradation of the hydrogel showed apparent gel erosion after the first three weeks, which is related to the IBU release rate: slow for the first three weeks and then fast. As a result, the total drug release after three and four weeks was 18 wt.% and 41 wt.%, respectively. However, in the first 24 hours, the amount of the drug released was 10 wt.%, suggesting that the IBU drug diffused from the surface hydrogel to the buffer solution. These show that PLA1750-PEG1750-PLA1750 hydrogel can be a potential IBU drug delivery candidate.

Assessing the biogeography of marine giant viruses in four oceanic transects.

Viruses of the phylum Nucleocytoviricota are ubiquitous in ocean waters and play important roles in shaping the dynamics of marine ecosystems. In this study, we leveraged the bioGEOTRACES metagenomic dataset collected across the Atlantic and Pacific Oceans to investigate the biogeography of these viruses in marine environments. We identified 330 viral genomes, including 212 in the order Imitervirales and 54 in the order Algavirales. We found that most viruses appeared to be prevalent in shallow waters (<150 m), and that viruses of the Mesomimiviridae (Imitervirales) and Prasinoviridae (Algavirales) are by far the most abundant and diverse groups in our survey. Five mesomimiviruses and one prasinovirus are particularly widespread in oligotrophic waters; annotation of these genomes revealed common stress response systems, photosynthesis-associated genes, and oxidative stress modulation genes that may be key to their broad distribution in the pelagic ocean. We identified a latitudinal pattern in viral diversity in one cruise that traversed the North and South Atlantic Ocean, with viral diversity peaking at high latitudes of the northern hemisphere. Community analyses revealed three distinct Nucleocytoviricota communities across latitudes, categorized by latitudinal distance towards the equator. Our results contribute to the understanding of the biogeography of these viruses in marine systems.

Assessing the biogeography of marine giant viruses in four oceanic transects.

Viruses of the phylum Nucleocytoviricota are ubiquitous in ocean waters and play important roles in shaping the dynamics of marine ecosystems. In this study, we leveraged the bioGEOTRACES metagenomic dataset collected across the Atlantic and Pacific Oceans to investigate the biogeography of these viruses in marine environments. We identified 330 viral genomes, including 212 in the order Imitervirales and 54 in the order Algavirales . We found that most viruses appeared to be prevalent in shallow waters (<150 meters), and that viruses of the Mesomimiviridae ( Imitervirales ) and Prasinoviridae ( Algavirales ) are by far the most abundant and diverse groups in our survey. Five mesomimiviruses and one prasinovirus are particularly widespread in oligotrophic waters; annotation of these genomes revealed common stress response systems, photosynthesis-associated genes, and oxidative stress modulation that may be key to their broad distribution in the pelagic ocean. We identified a latitudinal pattern in viral diversity in one cruise that traversed the North and South Atlantic Ocean, with viral diversity peaking at high latitudes of the northern hemisphere. Community analyses revealed three distinct Nucleocytoviricota communities across latitudes, categorized by latitudinal distance towards the equator. Our results contribute to the understanding of the biogeography of these viruses in marine systems.

Diversity and genomics of giant viruses in the North Pacific Subtropical Gyre.

Large double-stranded DNA viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are ubiquitous members of marine ecosystems that are important agents of mortality for eukaryotic plankton. Although giant viruses are known to be prevalent in marine systems, their activities in oligotrophic ocean waters remain unclear. Oligotrophic gyres constitute the majority of the ocean and assessing viral activities in these regions is therefore critical for understanding overall marine microbial processes. In this study, we generated 11 metagenome-assembled genomes (MAGs) of giant viruses from samples previously collected from Station ALOHA in the North Pacific Subtropical Gyre. Phylogenetic analyses revealed that they belong to the orders Imitervirales (n = 6), Algavirales (n = 4), and Pimascovirales (n = 1). Genome sizes ranged from ~119-574 kbp, and several of the genomes encoded predicted TCA cycle components, cytoskeletal proteins, collagen, rhodopsins, and proteins potentially involved in other cellular processes. Comparison with other marine metagenomes revealed that several have broad distribution across ocean basins and represent abundant viral constituents of pelagic surface waters. Our work sheds light on the diversity of giant viruses present in oligotrophic ocean waters across the globe.

MLK3 Regulates Inflammatory Response via Activation of AP-1 Pathway in HEK293 and RAW264.7 Cells.

Inflammation is a critically important barrier found in innate immunity. However, severe and sustained inflammatory conditions are regarded as causes of many different serious diseases, such as cancer, atherosclerosis, and diabetes. Although numerous studies have addressed how inflammatory responses proceed and what kinds of proteins and cells are involved, the exact mechanism and protein components regulating inflammatory reactions are not fully understood. In this paper, to determine the regulatory role of mixed lineage kinase 3 (MLK3), which functions as mitogen-activated protein kinase kinase kinase (MAP3K) in cancer cells in inflammatory response to macrophages, we employed an overexpression strategy with MLK3 in HEK293 cells and used its inhibitor URMC-099 in lipopolysaccharide (LPS)-treated RAW264.7 cells. It was found that overexpressed MLK3 increased the mRNA expression of inflammatory genes (COX-2, IL-6, and TNF-α) via the activation of AP-1, according to a luciferase assay carried out with AP-1-Luc. Overexpression of MLK3 also induced phosphorylation of MAPKK (MEK1/2, MKK3/6, and MKK4/7), MAPK (ERK, p38, and JNK), and AP-1 subunits (c-Jun, c-Fos, and FRA-1). Phosphorylation of MLK3 was also observed in RAW264.7 cells stimulated by LPS, Pam3CSK, and poly(I:C). Finally, inhibition of MLK3 by URMC-099 reduced the expression of COX-2 and CCL-12, phosphorylation of c-Jun, luciferase activity mediated by AP-1, and phosphorylation of MAPK in LPS-treated RAW264.7 cells. Taken together, our findings strongly suggest that MLK3 plays a central role in controlling AP-1-mediated inflammatory responses in macrophages and that this enzyme can serve as a target molecule for treating AP-1-mediated inflammatory diseases.