Small molecules that selectively modulate intracellular trafficking offer new strategies to tune innate immune signaling. Now, inhibitors called ENDOtollins are shown to target a key endosomal maturation checkpoint, attenuating inflammatory Toll-like receptor responses while preserving plasma membrane-initiated immunity.

Polyethylene terephthalate (PET) micro and nanoplastics (MNPs) are widely dispersed pollutants with significant environmental and health implications. Current preparation methods of representative secondary PET MNPs are limited. In this study, we developed a scalable, standardized ethylene glycol fragmentation (EGF)-based method to generate PET MNPs from three distinct plastic sources (commercial pellets, bottle-grade, and film-grade). PET samples were depolymerized using ethylene glycol and sodium carbonate under controlled thermal conditions. Two fractions (Ea and Eb) were collected and analyzed, fraction Ea consisted of covalently fragmented BHET-oligomer-based nanoplastics (∼200–375 nm), while fraction Eb comprised non-covalent BHET-based larger microplastic aggregates (∼1.2–1.9 µm). EGF method turned out to be more efficient than published trifluoroacetic acid (TFA) method giving mainly BHET-oligomer-based nanoplastics with higher yields and improved MNPs size control. MNPs generated by EGF showed a size range between 200 and 375 nm with good colloidal stability. In vitro assays showed that both type (covalent and non-covalent) of PET nanoplastics reduced THP-1 macrophage viability in a dose-dependent manner which was associated with IL-1β release, and triggered an M1-polarizing cytokine profile, thus indicating potential toxicity and proinflammatory activity. Our EGF-based synthesis platform enables controlled production of PET MNPs with source-dependent characteristics and biologically relevant behavior. These standardized MNPs and the THP-1 macrophages biosensor model provide useful tools for environmental toxicology research.

Background Acute Lung Injuries (ALI) are a severe consequence of influenza-induced cytokine storm that can cause respiratory failure and death. It has been demonstrated that Toll-like Receptor 4 (TLR4) is involved in cytokine storm and that TLR4 −/− mice are protected against ALI. Therefore, TLR4 is a prime target for protection against ALI. FP12 is a known TLR4 antagonist that reduces TLR4-dependent immune activation and it is a promising lead compound for the treatment of innate immunity related pathologies. Objectives We present here the preclinical development of FP12 as an anti-inflammatory lead compound acting on influenza-induced ALI. Methods In vitro: We pre-treated THP-1 cells with FP12 (10 μM) for 0.5 h, then exposed to LPS (100 ng/ml) for 0 to 16 h. In some experiments, cells were simultaneously incubated with FP12 and LPS, or FP12 was added 30 min after LPS. Cytokine levels were measured by Western blot and ELISA assays. In vivo: WT C57BL/6J mice were infected with mouse-adapted influenza virus (PR8). Two days after infection, mice received either vehicle, FP7 (200 µg/mouse), or FP12 (200 µg/mouse) once daily (Day 2 to Day 6). Mice were monitored daily for survival for 14 days. Data were collected through histological staining, qRT-PCR, and ELISA assay. Results FP12 treatment inhibited both LPS- and HMGB1-induced TLR4 intracellular pathways (MyD88 and TRIF) leading to significantly reduced levels of a variety of proinflammatory cytokines including Type I interferon (IFN-β), highlighting its effectiveness in controlling proinflammatory protein production and reducing inflammation. FP12 protected mice therapeutically from influenza virus-induced lethality and reduced both cytokine gene expression and High Mobility Group Box 1 (HMGB1) levels in the lungs as well as ALI. Conclusion FP12 can antagonize TLR4 activation in vitro and protects mice from severe influenza infection, most likely by reducing the TLR4-dependent cytokine storm mediated by danger-associated molecular patterns (DAMPs).

Toll-like receptors (TLRs), including TLR4, play a crucial role in innate immunity activation, and small molecular TLR4 activators (agonists) are in preclinical and clinical phase of development as vaccine adjuvant...

Vaccine adjuvants are critical to improve the immunogenicity, efficacy, and durability of vaccines; however, their development has lagged behind that of vaccine antigens. Monophosphoryl lipid A (MPLA), a clinically approved adjuvant that stimulates Toll-like receptor 4 (TLR4), faces manufacturing challenges due to its complex and long synthesis. With the aim of simplifying the structure of MPLA while retaining its biological activity, we developed monosaccharide-based molecules FP18 and FP20Rha that activate TLR4 signaling. Both TLR4 agonists induced robust antibody activity against the model antigen, ovalbumin. Here, we report the potential of these TLR4 agonists to enhance the protective efficacy of the well-characterized OprF antigen against P. aeruginosa infection. OprF adjuvanted with FP18 showed reduced bacterial loads in lungs and spleens, relative to antigen alone in an acute P. aeruginosa pneumonia model. FP18-adjuvanted OprF also enhanced the production of anti-OprF antibodies and stimulated IFNγ and TNF in CD4+ T cells, suggesting a Th1-skewed cellular immune response. These adjuvants have promise for accelerating the development of effective vaccines against P. aeruginosa and other infectious diseases.

Tannic acid is a natural polyphenol capable of strongly interacting with proteins, with good antioxidant and antibacterial properties. Thus, tannic acid (TA) or oxidized tannic acid (oxTA) may be used as cross-linking agents in the development of reinforced and fully protein-based superabsorbent materials (SAMs). oxTA was produced so that reactive quinone groups were generated, which are expected to increase its reactivity. In this study, porcine plasma protein (PPP) and glycerol (gly) were used in a 50/50 PPP/gly ratio to obtain SAMs through twin screw mixing and injection molding. The results showed that both TA and oxTA increased the storage modulus and the loss tangent of blends and bioplastics due to the physical interactions established between TA or oxTA and PPP. The mechanical properties, particularly the Young's modulus and tensile strength, were generally enhanced as well. Water absorption was strongly influenced by the addition of TA, resulting in a decrease in the amount of water absorbed. However, samples containing oxTA resulted in a greater water absorption capacity, retaining a higher proportion of the superabsorbent properties of the reference composition. Moreover, systems containing oxTA generally possess better mechanical properties than those of equivalent TA formulations, especially those containing 5 % and 10 % oxTA.

The activation of TLR4 by endogenous damage or danger-associated molecular patterns (DAMPs) suggested the use of TLR4 antagonists to target acute and chronic inflammatory diseases. In recent years, hundreds of natural compounds (NCs) have been screened for their activity as TLR4 antagonists. However, the direct interaction with TLR4 or TLR4/MD-2 dimer has not been proven for most of the natural molecules, and their mechanism of action has been only partially investigated. We review the recent literature on NCs active as TLR4 antagonists, analyzing the limitations and selecting among all candidates the compounds presenting new scaffolds desirable for drug development. After selecting a collection of representative hit structures, we also present novel docking calculations on TLR4/MD-2. Our analysis allowed the detection of common binding motifs in the receptor and the proposal of a structure-activity relationship from the ligands, enabling the discovery of new ligand scaffolds and pharmacophores for further development of drug hits.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

There is a growing need to find novel, sustainable solutions for elastomer reinforcement. Many biomass polymers have been used for this purpose, including cellulose, starch, lignin and protein. Among these, proteins are an under-explored solution, although they are promising materials due to their abundance in agricultural and animal by-products and the multifunctional properties they can impart to composites. Proteins are naturally occurring components of Hevea Brasiliensis natural rubber, where many of its peculiar properties have been attributed to these biopolymers, such as strain-induced crystallization, improved resistance to thermo-oxidative aging and vulcanization accelerating effects. The production of ‘artificial’ rubber/protein composites using these biopolymers offers other advantages such as low cost, lightweight, biodegradability and high availability. This review focuses on wheat gluten, corn zein, keratin and collagen. The incorporation of such materials into elastomeric matrices is challenging due to their hydrophilicity; therefore, dispersion and interfacial adhesion need to be improved to obtain composites with improved properties. In this sense, several strategies have been addressed in the literature to solve this problem, including rubber and/or protein functionalization and different compounding techniques. The aim of this review is to describe and compare these strategies in order to provide a toolbox for optimal composite fabrication.

In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.

In this study, we formulated an alternative to AS01b by combining FP20, a synthetic TLR4 agonist, and QS21v, a minimal saponin adjuvant, aiming to improve the vaccine efficacy and stability. The phase transition temperature of FP20 was determined by using differential scanning calorimetry to be 43.9 °C, providing a foundation for the formulation process. The coformulation was prepared using a dry film method for even adjuvant distribution. Characterization by dynamic light scattering and nanoparticle tracking analysis revealed a uniform particle size distribution of ∼120 nm. Cryogenic electron microscopy (CryoEM) revealed nanosized interactions between FP20 and QS21v, forming stable structures that likely enhanced the antigen presentation and immune activation. These physicochemical properties contributed to a robust in vivo synergy, where the coformulation elicited significantly higher antigen-specific antibody titers compared to individual adjuvants. These findings suggest that the FP20+QS21v coformulation provides a potent, stable, and safer alternative to traditional adjuvants, enhancing both vaccine efficacy and immunogenicity.

BACKGROUND:The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS:Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS:As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS:The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Vaccines are one of the greatest achievements of modern medicine. Due to their safer profile, the latest investigations usually focus on subunit vaccines. However, the active component often needs to be coupled with an adjuvant to be effective and properly trigger an immune response. We are developing a new synthetic monosaccharide-based TLR4 agonist, such as glucosamine-derived compounds FP18 and FP20, as a potential vaccine adjuvant. In this study, we present a new FP20 derivative, FP20Hmp, with a hydroxylated ester linked to the glucosamine core. We show that the modification introduced improves the activity of the adjuvant and its solubility. This study presents the synthesis of FP20Hmp, its in vitro characterization, and in vivo activity while coupled with the ovalbumin antigen or in formulation with an enterococcal antigen. We show that FP20Hmp enables increased production of antigen-specific antibodies that bind to the whole bacterium.

We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.

Cancer stem cells (CSCs) have drawn much attention as important tumour-initiating cells that may also be crucial for recurrence after chemotherapy. Although the activity of CSCs in various forms of cancer is complex and yet to be fully elucidated, opportunities for therapies targeting CSCs exist. CSCs are molecularly distinct from bulk tumour cells, so they can be targeted by exploiting their signature molecular pathways. Inhibiting stemness has the potential to reduce the risk posed by CSCs by limiting or eliminating their capacity for tumorigenesis, proliferation, metastasis, and recurrence. Here, we briefly described the role of CSCs in tumour biology, the mechanisms involved in CSC therapy resistance, and the role of the gut microbiota in cancer development and treatment, to then review and discuss the current advances in the discovery of microbiota-derived natural compounds targeting CSCs. Collectively, our overview suggests that dietary intervention, toward the production of those identified microbial metabolites capable of suppressing CSC properties, is a promising approach to support standard chemotherapy.

We developed synthetic glycophospholipids based on a glucosamine core (FP compounds) with potent and selective activity in stimulating Toll-Like Receptor 4 (TLR4) as agonists. These compounds have activity and toxicity profiles similar to the clinically approved adjuvant monophosphoryl lipid A (MPLA), included in several vaccine formulations, and are now in the preclinical phase of development as vaccine adjuvants in collaboration with Croda International PLC. FP compound synthesis is shorter and less expensive than MPLA preparation but presents challenges due to the use of toxic solvents and hazardous intermediates. In this paper we describe the optimization of FP compound synthesis. The use of regio- and chemoselective reactions allowed us to reduce the number of synthesis steps and improve process scalability, overall yield, safety, and Process Mass Intensity (PMI), thus paving the way to the industrial scale-up of the process.

Fluorescent chemical probes are used nowadays as a chemical resource to study the physiology and pharmacology of several important endogenous receptors. Different fluorescent groups have been coupled with known ligands of these receptors, allowing the visualization of their localization and trafficking. One of the most important molecular players of innate immunity and inflammation are the Toll-Like Receptors (TLRs). These Pattern-Recognition Receptors (PRR) have as natural ligands microbial-derived pathogen-associated molecular patterns (PAMPs) and also endogenous molecules called danger-associated molecular patterns (DAMPs). These ligands activate TLRs to start a response that will determine the host's protection and overall cell survival but can also lead to chronic inflammation and autoimmune syndromes. TLRs action is tightly related to their subcellular localization and trafficking. Understanding this trafficking phenomenon can enlighten critical molecular pathways that might allow to decipher the causes of different diseases. In this chapter, the study of function, localization and trafficking of TLRs through the use of chemical probes will be discussed. Furthermore, an example protocol of the use of fluorescent chemical probes to study TLR4 trafficking using high-content analysis will be described.

Lipopolysaccharide (LPS) mimicry leading to toll-like receptor 4 (TLR4) active compounds has been so far based mainly on reproducing the lipid A portion of LPS. Our work led to a series of structurally simplified synthetic TLR4 agonists in preclinical development as vaccine adjuvants called FPs. FPs bind MD2/TLR4 similarly to lipid A, inserting the lipid chains in the MD2 lipophilic cavity. A strategy to improve FPs' target affinity is introducing a monosaccharide unit in C6, mimicking the first sugar of the LPS core. We therefore designed a panel of FP derivatives bearing different monosaccharides in C6. We report here the synthesis and optimization of FPs' C6 glycosylation, which presented unique challenges and limitations. The biological activity of glycosylated FP compounds was preliminarily assessed in vitro in HEK-Blue cells. The new molecules showed a higher potency in stimulating TLR4 activation when compared to the parent molecule while maintaining TLR4 selectivity.