Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages.

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and 'synthetic peptide antibodies' were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Degradation of Residual Herbicide Atrazine in Agri-Food and Washing Water.

Atrazine, an herbicide used to control grassy and broadleaf weed, has become an essential part of agricultural crop protection tools. It is widely sprayed on corn, sorghum and sugar cane, with the attendant problems of its residues in agri-food and washing water. If ingested into humans, this residual atrazine can cause reproductive harm, developmental toxicity and carcinogenicity. It is therefore important to find clean and economical degradation processes for atrazine. In recent years, many physical, chemical and biological methods have been proposed to remove atrazine from the aquatic environment. This review introduces the research works of atrazine degradation in aqueous solutions by method classification. These methods are then compared by their advantages, disadvantages, and different degradation pathways of atrazine. Moreover, the existing toxicological experimental data for atrazine and its metabolites are summarized. Finally, the review concludes with directions for future research and major challenges to be addressed.

Molecularly imprinted polymer nanogels targeting the HAV motif in cadherins inhibit cell-cell adhesion and migration.

Cadherins are cell-surface proteins that mediate cell-cell adhesion. By regulating their grip formation and strength, cadherins play a pivotal role during normal tissue morphogenesis and homeostasis of multicellular organisms. However, their dysfunction is associated with cell migration and proliferation, cancer progression and metastasis. The conserved amino acid sequence His-Ala-Val (HAV) in the extracellular domain of cadherins is implicated in cadherin-mediated adhesion and migration. Antagonists of cadherin adhesion such as monoclonal antibodies and small molecule inhibitors based on HAV peptides, are of high therapeutic value in cancer treatment. However, antibodies are not stable outside their natural environment and are expensive to produce, while peptides have certain limitations as a drug as they are prone to proteolysis. Herein, we propose as alternative, a synthetic antibody based on molecularly imprinted polymer nanogels (MIP-NGs) to target the HAV domain. The MIP-NGs are biocompatible, have high affinity for N-cadherin and inhibit cell adhesion and migration of human cervical adenocarcinoma (HeLa) cells, as demonstrated by cell aggregation and Matrigel invasion assays, respectively. The emergence of MIPs as therapeutics for fighting cancer is still in its infancy and this novel demonstration reinforces the fact that they have a rightful place in cancer treatment.

A water-soluble polysaccharide from Anethum graveolens seeds: Structural characterization, antioxidant activity and potential use as meat preservative.

A novel water-soluble polysaccharide named AGP1 was successfully isolated from seeds of Anethum graveolens by hot water extraction and further purified by DEAE-Sepharose chromatography. AGP1 has a relative molecular weight of 2.1 104 Da determined by Ultra-high-performance liquid chromatography (UHPLC). The AGP1 characterization was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction. Results showed that AGP1 was mainly composed of glucose, galactose, mannose and arabinose in a molar percent of 54.3, 23.8, 14.7 and 7.2, respectively. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that AGP1 has good thermal stability until 275 °C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential. The addition of the AGP1, particularly at 0.3% (w/w), in turkey sausages instead of ascorbic acid, as preservative, reduced the lipid peroxidation, preserved the pH and color and improved the bacterial stability during cold storage at 4 °C for 12 days. Overall, the results showed that the AGP1 deserves to be developed as functional and bioactive components for the food and nutraceutical industries.

Chemical Antibody Mimics Inhibit Cadherin-Mediated Cell-Cell Adhesion: A Promising Strategy for Cancer Therapy.

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy.

Evaluation of performance and validity limits of gas chromatography electron ionisation with Orbitrap detection for fatty acid methyl ester analyses.

RATIONALE: While the GC-Orbitrap, marketed in 2015, represents a technological breakthrough in terms of sensitivity, resolution and mass stability, many studies have reported ion ratio modification in mass spectra using the standard 70 eV electron ionisation. METHODS: We studied the influence of the acquisition and sample parameters leading to these modifications on fatty acid methyl esters (FAMEs). RESULTS: FAMEs showed that these variations in relative intensities of ions were related to the acquisition parameters such as the mass range and the offset values of the C-TRAP, but also directly related to the column concentration of the sample, and especially that it was molecule-dependent. Advantageously, it is possible to use this feature to promote the molecular ions of FAMEs sometimes not present in a spectrum under electron ionisation at 70 eV. CONCLUSIONS: The 70 eV electron ionisation mass spectra from the GC-Orbitrap were clearly molecule-dependent and could be due to metastable ions during storage states in the C-TRAP.

Composition, antibacterial and antioxidant activities of Pimpinella saxifraga essential oil and application to cheese preservation as coating additive.

The effect of Pimpinella saxifraga essential oil (PSEO) addition (1-3%) in sodium alginate coating on the bacterial and oxidative stability of cheese was studied during refrigerated storage. The GC-HRMS analysis of PSEO showed that anethole, pseudoisoeugenol and p-anisaldehyde were the main components. The PSEO exhibited strong in vitro DPPH radical scavenging activity (IC50 = 6.81 µg/mL), ß-carotene bleaching inhibition (IC50 = 206 µg/mL), ferric reducing power (EC50 = 35.20 µg/mL), total antioxidant activity (213.96 ±â€¯11.12 µmol/mL α-tocopherol equivalent) and notable DNA protection potential. Additionally, PSEO displayed potent antibacterial activity against 3 Gram-positive and 3 Gram-negative bacteria (MICs = 0.78-3.12 mg/mL). The acute toxicity of PSEO was determined using mice model (LD50 = 976.2 mg/kg). The enrichment of sodium alginate coating with PSEO, particularly at 3%, improved cheese preservation by reducing the weight loss, preserving the pH and color and enhancing oxidative and bacterial stability without unpleased flavor for consumers.

Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic Antibodies for Immunoprotection against HIV.

We describe the preparation and characterization of synthetic antibodies based on molecularly imprinted polymer nanoparticles (MIP-NPs) for the recognition and binding of the highly conserved and specific peptide motif SWSNKS (3S), an epitope of the envelope glycoprotein 41 (gp41) of human immunodeficiency virus type 1 (HIV-1). This motif is implicated in the decline of CD4+ T cells and leads to the deterioration of the immune system during HIV infection. Therefore, the development of MIP-NPs that can target and block the 3S peptide to prevent subsequent cascade interactions directed toward the killing of CD4+ T cells is of prime importance. Because most antibodies recognize their protein antigen via a conformational or structured epitope (as opposed to a linear epitope commonly used for molecular imprinting), we employed protein molecular modeling to design our template epitope so that it mimics the three-dimensional structure fold of 3S in gp41. The resulting template peptide corresponds to a cyclic structure composed of CGSWSNKSC, with the 3S motif well orientated for imprinting. MIP-NPs with a size of 65 nm were obtained by solid-phase synthesis and were water-soluble. They were prepared by a judicious combination of multiple functional monomers affording hydrogen bonding, ionic, π-π, and hydrophobic interactions, conferring high affinity and selectivity toward both the cyclic peptide and the whole gp41 protein. These results suggest that our MIPs could potentially be used for blocking the function of the 3S motif on the virus.

A gas chromatography full scan high resolution Orbitrap mass spectrometry method for separation and characterization of 3-hydroxymethyl pyridine ester of fatty acids at low levels.

Fatty acid methyl esters (FAMEs), which are commonly used to characterize lipids, have several limitations to conclude on many structures. 3-Pyridylcarbinol esters (3-PCE) are used to characterize fatty acid structures [1], in particular, to identify ring and double bond positions on the carbon chain. Chromatographic separation of these esters is complex due to their polarity and high boiling points. In this study, we used a column with high resolutive power based on ionic liquids to increase the separation quality in gas chromatography (GC). In addition, we used a high-resolution detector (Orbitrap) to limit non-specific signals and improve the detection limits. This detector could be used with a mass filter at 5 ppm for the rapid determination of 3-PCE from its characteristic ions (m/z = 108.0441 and 92.0495). This filter allowed the identification of derivative fatty acids with good sensibility. Thus, it was possible to characterize 3-PCE by measuring the exact fragment masses to confirm structures such as C19:2n12cycloΔ9.

Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

Betanin-Enriched Red Beetroot (Beta vulgaris L.) Extract Induces Apoptosis and Autophagic Cell Death in MCF-7 Cells.

Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines.

We developed a new biological model to mimic the organ-organ interactions between the intestine and the liver. We coupled polycarbonate cell culture inserts and microfluidic biochips in an integrated fluidic platform allowing dynamic co-cultures (called IIDMP for Integrated Insert in a Dynamic Microfluidic Platform). The intestinal compartment was simulated using Caco-2 TC7 cells and the liver one by HepG2 C3A. We showed that Caco-2 TC7 viability, barrier integrity and functionality (assessed by paracellular and active transport), were not altered during co-cultures in the bioreactor in comparison with the conventional insert Petri cultures. In parallel, the viability and metabolism of the HepG2 C3A cells were maintained in the microfluidic biochips. Then, as proof of concept, we used the bioreactor to follow the transport of phenacetin through the intestinal barrier and its metabolism into paracetamol by the CYP1A of the HepG2 C3A cells. Our results demonstrated the performance of this bioreactor with cell co-cultures compared to static co-culture controls in which weak biotransformation into paracetamol was detected. Our study illustrated the interest of such a bioreactor combining the advantages of a cell culture barrier and of liver microfluidic cultures in a common framework for in vitro studies.

The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology.

Integrated proteomic and transcriptomic investigation of the acetaminophen toxicity in liver microfluidic biochip.

Microfluidic bioartificial organs allow the reproduction of in vivo-like properties such as cell culture in a 3D dynamical micro environment. In this work, we established a method and a protocol for performing a toxicogenomic analysis of HepG2/C3A cultivated in a microfluidic biochip. Transcriptomic and proteomic analyses have shown the induction of the NRF2 pathway and the related drug metabolism pathways when the HepG2/C3A cells were cultivated in the biochip. The induction of those pathways in the biochip enhanced the metabolism of the N-acetyl-p-aminophenol drug (acetaminophen-APAP) when compared to Petri cultures. Thus, we observed 50% growth inhibition of cell proliferation at 1 mM in the biochip, which appeared similar to human plasmatic toxic concentrations reported at 2 mM. The metabolic signature of APAP toxicity in the biochip showed similar biomarkers as those reported in vivo, such as the calcium homeostasis, lipid metabolism and reorganization of the cytoskeleton, at the transcriptome and proteome levels (which was not the case in Petri dishes). These results demonstrate a specific molecular signature for acetaminophen at transcriptomic and proteomic levels closed to situations found in vivo. Interestingly, a common component of the signature of the APAP molecule was identified in Petri and biochip cultures via the perturbations of the DNA replication and cell cycle. These findings provide an important insight into the use of microfluidic biochips as new tools in biomarker research in pharmaceutical drug studies and predictive toxicity investigations.