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Establishment of novel, highly productive, and sustainable processes for the production of industrially important compounds is becoming a growing area of research. Due to non-volatility, inflammability, great thermal, chemical and electrochemical stability and also recyclability, ionic liquids are extensively studied as possible green replacement for widely used conventional molecular solvents. Due to the extremely large number of possible chemical structures of ionic liquids, the ability to design ionic liquids for specific applications makes them unique solvents in electrochemistry, analytical chemistry, organic synthesis and (bio)catalysis, separation processes, as well as functional fluids (lubricants, heat transfer fluids, corrosion inhibitors etc.). This paper presents a review of the scientific and technical literature related to ionic liquids, their basic properties, preparation and application, as well as the challenges of their application on an industrial scale.

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Natural deep eutectic solvents (NADES) may be considered 'designer solvents' due to their numerous structural variations and the possibility of tailoring their physicochemical properties. Prior to their industrial application, characterization of NADES is essential, including determination of their physicochemical properties, cytotoxicity, and antioxidative activity. The most important physicochemical properties of eight prepared NADES (choline chloride:malic acid, proline:malic acid, choline chloride:proline:malic acid, betaine:malic acid, malic acid:glucose, malic acid:glucose:glycerol, choline chloride: citric acid, and betaine:citric acid) were measured as functions of temperature and water content. In general, the structure of prepared NADES greatly influences their physical properties, which could be successfully modified and adjusted by addition of water. All tested NADES were absolutely benign and noncorrosive for investigated steel X6CrNiTi18-10. Furthermore, cytotoxicity of prepared solvents was assessed toward three human cell lines (HEK-293T, HeLa, and MCF-7 cells), and antioxidative activity was measured by the Oxygen Radical Absorbance Capacity (ORAC) method. With regard to cell viability, all tested NADES containing carboxylic acid could be classified as practically harmless and considered environmentally safe. The ORAC values indicated that the tested NADES displayed antioxidative activity. [ABSTRACT FROM AUTHOR]

Copyright of Kemija u Industriji is the property of Croatian Society of Chemical Engineers and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Proline cis-trans conformational isomerization is a mechanism that affects different types of protein functions and behaviors. Using analytical characterization, structural analysis, and molecular dynamics simulations, we studied the causes of an aberrant two-peak size-exclusion chromatography profile observed for a trispecific anti-HIV antibody. We found that proline isomerization in the tyrosine-proline-proline (YPP) motif in the heavy chain complementarity-determining region (CDR)3 domain of one of the antibody arms (10e8v4) was a component of this profile. The pH effect on the conformational equilibrium that led to these two populations was presumably caused by a histidine residue (H147) in the light chain that is in direct contact with the YPP motif. Finally, we demonstrated that, due to chemical equilibrium between the cis and trans proline conformers, the antigen-binding potency of the trispecific anti-HIV antibody was not significantly affected in spite of a potential structural clash of 10e8v4 YPtransPtrans conformers with the membrane-proximal ectodomain region epitope in the GP41 antigen. Altogether, these results reveal at mechanistic and molecular levels the effect of proline isomerization in the CDR on the antibody binding and analytical profiles, and support further development of the trispecific anti-HIV antibody.

Broccoli is rich in biologically active compounds, especially polyphenols and glucosinolates, known for their health benefits. Traditional extraction methods have limitations, leading to a shift towards using natural deep eutectic solvents (NADESs) to create high-quality extracts with enhanced biological activity. This study focuses on preparing broccoli extracts in NADES, enriched with polyphenols and glucosinolates, without additional purification steps. Using the COSMOtherm software, the solubility of polyphenols and glucosinolates in NADESs was predicted, and five biocompatible betaine-based NADESs were prepared with glucose (B:Glc 1:1 and B:Glc 5:2 ), sucrose (B:Suc), glycerol (B:Gly), and malic acid (B:MA) as hydrogen bond donors. The resulting extracts were assessed for total polyphenol and glucosinolate content, along with antioxidant capacity, using the ORAC assay. The results demonstrated that NADES extracts contained higher polyphenol content and exhibited enhanced antioxidant effects compared to the reference ethanol extract, with B:Glc 1:1 extract showing the highest performance among all the extracts tested. On the other hand, the extract based on B:MA exhibited nearly six times higher total glucosinolate content compared to the ethanol extract. Additionally, polyphenols and glucosinolates were generally more stable in NADES extracts than in the reference solvent. Finally, the B:Glc 1:1 extract, identified as optimal in terms of polyphenol and glucosinolate content and stability, exhibited mild stimulation of HaCaT cells growth and facilitated the wound-healing process. Through green chemistry parameter calculations, we demonstrated that the extraction of broccoli bioactives using B:Glc 1:1 can be considered sustainable, underscoring the potential of NADESs for producing ready-to-use plant extracts.

In the context of a circular economy, this study explores the valorization of blueberry pomace (BP) as a source of bioactive compounds using sustainable extraction methods. Microwave-assisted extraction (MAE) and microwave-assisted subcritical water extraction (MASWE) were employed to obtain two distinct fractions: MAE 1° and MASWE 2°. The first extract, MAE 1°, obtained at 80 °C, had a high total anthocyanin content (21.96 mg Cya-3-glu /g extract ), making it suitable as a natural pigment. Additionally, MAE 1° exhibited significant enzyme inhibition, particularly against α-amylase and β-glucosidase, suggesting potential anti-diabetic and anti-viral applications. The second extract, MASWE 2°, obtained at 150 °C, contained a higher total phenolic content (211.73 mg GAE /g extract ) and demonstrated stronger antioxidant activity. MASWE 2° showed greater inhibition of acetylcholinesterase and tyrosinase, indicating its potential for use in Alzheimer's treatment, skincare, or as a food preservative. MASWE 2° exhibited cytotoxicity against HeLa cells and effectively mitigated H 2 O 2 -induced oxidative stress in HaCat cells, with MAE 1° showing similar but less pronounced effects. A tested formulation combining MAE 1° and MASWE 2° extracts in a 3:2 ratio effectively enhanced anthocyanin stability, demonstrating its potential as a heat-stable pigment. The extract characteristics were compared with a conventional method (MeOH-HCl in reflux condition), and the protocol's sustainability was assessed using several green metric tools, which provided insights into its environmental impact and efficiency.

Pomegranate ( Punica granatum L.) is well known for its high content of bioactives, including polyphenols, flavonoids, and tannins, which have been shown to exhibit a wide range of biological activities, such as antioxidant, antimicrobial, and anticancer effects. It is worth noting that the majority of these molecules are found in the peels, which are usually disposed of after processing, causing a significant amount of waste, amounting to more than 3.6 million t/y. This work investigates microwave-assisted extraction (MAE) in water for the recovery of antioxidants from pomegranate peels (PP), including the optimisation of temperature and extraction times. The total phenolic, anthocyanin, flavonoid, and tannin contents of the recovered extracts were determined, as well as their antioxidant activities, which were found to be 356.35 mgGAE/gExtr, 303.97 µgCy3G/gExtr, 37.28 mgQE/gExtr, 56.48 mgGAE/gExtr, and 5.72 mmolTE/gExtr, respectively (according to the adopted reference). All results were compared with those obtained using a conventional protocol. In addition, the potential for water recycling by means of downstream nanofiltration in optimised MAE was investigated, leading to overall water reuse of approx. 75%. Power consumption (20.92 W/mgGAE) and common green metrics, Reaction Mass Efficiency (RME), E-Factor, and the Process Mass Intensiti/efficiency (PMI, PME), were considered in evaluating the proposed PP valorisation strategy. Finally, the biological activities of the main products were assessed. The antimicrobial properties of the PP extracts against three Gram-positive and three Gram-negative bacteria and their antiproliferative activity towards human cancer cells were tested. S. aureus bacteria was the most susceptible to the PP extracts. All tested products displayed antiproliferative activity against HeLa cells when higher concentrations were tested, with D-PP/NF (obtained from dried PP and sequential nanofiltration) being the most effective. This result was also confirmed via clonogenic analysis, which generally indicated the possible anti-cancer activity of pomegranate peel extracts obtained using this green approach.

The aim of this work was to develop a simple and easy-to-apply model to predict the pH values of deep eutectic solvents (DESs) over a wide range of pH values that can be used in daily work. For this purpose, the pH values of 38 different DESs were measured (ranging from 0.36 to 9.31) and mathematically interpreted. To develop mathematical models, DESs were first numerically described using σ profiles generated with the COSMOtherm software. After the DESs’ description, the following models were used: (i) multiple linear regression (MLR), (ii) piecewise linear regression (PLR), and (iii) artificial neural networks (ANNs) to link the experimental values with the descriptors. Both PLR and ANN were found to be applicable to predict the pH values of DESs with a very high goodness of fit (R2independent validation > 0.8600). Due to the good mathematical correlation of the experimental and predicted values, the σ profile generated with COSMOtherm could be used as a DES molecular descriptor for the prediction of their pH values.

The concept of peptidomimetics is based on structural modifications of natural peptides that aim not only to mimic their 3D shape and biological function, but also to reduce their limitations. The peptidomimetic approach is used in medicinal chemistry to develop drug-like compounds that are more active and selective than natural peptides and have fewer side effects. One of the synthetic strategies for obtaining peptidomimetics involves mimicking peptide α-helices, β-sheets or turns. Turns are usually located on the protein surface where they interact with various receptors and are therefore involved in numerous biological events. Among the various synthetic tools for turn mimetic design reported so far, our group uses an approach based on the insertion of different ferrocene templates into the peptide backbone that both induce turn formation and reduce conformational flexibility. Here, we conjugated methyl 1'-aminoferrocene-carboxylate with homo- and heterochiral Pro-Ala dipeptides to investigate the turn formation potential and antiproliferative properties of the resulting peptidomimetics 2 - 5 . Detailed spectroscopic (IR, NMR, CD), X-ray and DFT studies showed that the heterochiral conjugates 2 and 3 were more suitable for the formation of β-turns. Cell viability study, clonogenic assay and cell death analysis showed the highest biological potential of homochiral peptide 4 .

The aim of this work is to develop an industrially suitable process for the sustainable waste disposal in wine production. The proposed process involves the development of an environmentally friendly method for the isolation of biologically active compounds from Graševina grape pomace according to the green extraction principles, in order to obtain a ready-to-use extract. In this process, deep eutectic solvents (DES) were used as extraction solvents. Aiming to save time in selecting the optimal DES that would provide the most efficient Graševina pomace polyphenols extraction, the user-friendly software COSMOtherm was used and 45 DES were screened. Moreover, the prepared extracts were chemically and biologically characterized to confirm their safety for human application. Computational and experimental results proved the applicability of COSMOtherm in the selection of the optimal DES for the environmentally friendly preparation of the ready-to-use extract from Graševina grape pomace with expected application in the cosmetic industry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for one of the worst pandemics in modern history. Several prevention and treatment strategies have been designed and evaluated in recent months either through the repurposing of existing treatments or the development of new drugs and vaccines. In this study, we show that L-carnitine tartrate supplementation in humans and rodents led to significant decreases of key host dependency factors, notably angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and Furin, which are responsible for viral attachment, viral spike S-protein cleavage, and priming for viral fusion and entry. Interestingly, pre-treatment of Calu-3, human lung epithelial cells, with L-carnitine tartrate led to a significant and dose-dependent inhibition of the infection by SARS-CoV-2. Infection inhibition coincided with a significant decrease in ACE2 mRNA expression levels. These data suggest that L-carnitine tartrate should be tested with appropriate trials in humans for the possibility to limit SARS-CoV-2 infection.

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC 50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity., Competing Interests: Conflict of interest—All authors are or were employees of Sanofi., (© 2020 Stefano et al.)

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

Interest in bioactive phytochemicals and sustainable processes is the driving force behind this study on two novel green extraction methods for the recovery of anthocyanins from the residues of blueberry processing. Five natural deep eutectic solvents (NADES) have been tested for anthocyanin extraction. Acidified hydroalcoholic solutions were used as benchmarks and the shelf life of eutectic systems was monitored. The most promising NADES was tested in microwave (MAE)- and ultrasound-assisted extractions (UAEs), and Peleg's kinetic model was used. Both the enabling technologies provided performance that was superior to that of conventional extraction. MAE and UAE yielded up to 25.83 and 21.18 mg/g matrix of total anthocyanin content, respectively, after 15 and 30 min. Moreover, a preliminary test for extract concentration and NADES recycling was performed using resin adsorption. Finally, the antiproliferative activity of the extracts was determined by a CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay, the so-called MTS assay, on human tumour HeLa cells and human skin HaCaT cells. Nonconventional extracts exhibited strong antiproliferative activity that was much greater than that of their conventionally extracted analogues. Flow cytometry was used to evaluate cell-death type, and apoptosis was found to be the primary cause of tumour cell death. The presented study demonstrates that the implementation of enabling extraction technologies and green solvents can produce an antiproliferative agent from a food industry byproduct.

Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics., Competing Interests: BC, TD, LBS, MG, IA AS, FS, KR & TB are affiliated to SANOFI R&D JM, PAL, WH & KC are affiliated to LIGAND PHARMACEUTICAL INC. YA is affiliated to CARTERRA INC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Monoclonal antibodies (mAbs) are among the fastest growing and most effective therapies for myriad diseases. Multispecific antibodies are an emerging class of novel therapeutics that can target more than one tumor- or immune-associated modulators per molecule. The combination of different binding affinities and target classes, such as soluble or membrane-bound antigens, within multispecific antibodies confers unique pharmacokinetic (PK) properties. Numerous factors affect an antibody's PK, with affinity to the neonatal Fc receptor (FcRn) a key determinant of half-life. Recent work has demonstrated the potential for humanized FcRn transgenic mice to predict the PK of mAbs in humans. However, such work has not been extended to multispecific antibodies. We engineered mAbs and multispecific antibodies with various Fc modifications to enhance antibody performance. PK analyses in humanized FcRn transgenic mouse (homozygous Tg32 and Tg276) and non-human primate (NHP) models showed that FcRn-binding mutations improved the plasma half-lives of the engineered mAbs and multispecific antibodies, while glycan engineering to eliminate effector function did not affect the PK compared with wild-type controls. Furthermore, results suggest that the homozygous Tg32 mouse model can replace NHP models to differentiate PK of variants during lead optimization, not only for wild-type mAbs but also for Fc-engineered mAbs and multispecific antibodies. This Tg32-mouse model would enable prediction of half-life and linear clearance of mAbs and multispecific antibodies in NHPs to guide the design of further pharmacology/safety studies in this species. The allometric exponent for clearance scaling from Tg32 mice to NHPs was estimated to be 0.91 for all antibodies.

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.

Over the past three decades a growing awareness of environmental protection prompted the development of so-called green and sustainable technologies. Therefore, academic and wider community intensively explores new chemicals and safer, more energy efficient processes based on a rational compromise between economic, social, and environmental requirements. Due to low volatility and stability, ionic liquids emerged as a potential replacement for traditional volatile and harmful organic solvents. Various studies have been carried out to validate the green character of ionic liquids, whereby data published suggest that these compounds, due to their relatively high toxicity and poor biodegradability, could have an extremely negative impact on the environment. This paper presents the current knowledge on the toxicity of ionic liquids, with a special emphasis on the mechanisms by which this group of compounds causes changes in the morphology and physiology of organisms at different organisational levels of the ecosystem.

Three β-cyclocitral-derived halolactones, which exhibit antifeedant activity towards storage product pests, were subjected to microbial transformation processes. Among the thirty tested strains of filamentous fungi and yeast, the most effective biocatalysts were Absidia cylindrospora AM336, Mortierella isabellina AM212 and Mortierella vinaceae AM149. As a result of regio- and enantioselective hydroxylation four new oxygenated derivatives were obtained. Regardless of the biocatalyst applied, the δ-iodo- and δ-bromo-γ-lactones were hydroxylated in an inactivated position C-5 of cyclohexane ring. The analogous transformation of chlorolactone was observed in Mortierella isabellina AM212 culture but in the case of two other biocatalysts the hydroxy group was introduced at C-3 position. All obtained hydroxylactones were enantiomerically pure (ee = 100%) or enriched (ee = 50%). The highest enantioselectivity of hydroxylation was observed for M. isabellina AM212. The cytotoxic activity of halolactones was also examined by WST-1 assay wherein tested compounds did not exhibit significant effect on the viability of tumor HeLa and normal CHO-K1 cells.

Background: Various studies have shown that infants under the age of 6 months are especially vulnerable for complications due to influenza. Currently there are no vaccines licensed for use in this age group. Vaccination of pregnant women during the last trimester, recommended by the WHO as protective measure for this vulnerable female population, may provide protection of newborns at this early age. Although it has been observed that maternal vaccination can passively transfer protection, maternal antibodies could possibly also interfere with subsequent active vaccination of the offspring., Methods: Using a mouse model, we evaluated in depth the ability of maternal influenza vaccination to protect offspring and the effect of maternal immunization on the subsequent influenza vaccination of the offspring. By varying the regimen of maternal immunization we explored the impact of different levels of maternal antibodies on the longevity of these antibodies in their progeny. We subsequently assessed to what extent maternal antibodies can mediate direct protection against influenza in their offspring, and whether these antibodies interfere with protection induced by active vaccination of the offspring., Results: The number of immunizations of pregnant mice correlates to the level and longevity of maternal antibodies in the offspring. When these antibodies are present at time of influenza challenge they protect offspring against lethal influenza challenge, even in the absence of detectable HAI titers. Moreover, no detectable interference of passively-transferred maternal antibodies on the subsequent vaccination of the offspring was observed., Conclusion: In the absence of a licensed influenza vaccine for young children, vaccination of pregnant women is a promising measure to provide protection of young infants against severe influenza infection.

Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs.

With the advent of ionic liquids, much was expected concerning their applicability as an alternative to organic solvents in the chemical technology and biotechnology fields. However, the most studied and commonly used ionic liquids based on imidazolium and pyridinium were found not to be as environmentally friendly as it was first expected. Therefore, a new generation of alternative solvents named natural ionic liquids and deep eutectic solvents, composed of natural and/or renewable compounds, have come into focus in recent years. Since the number of newly synthesized chemicals increases yearly, simple and reliable methods for their ecotoxicological assessment are necessary. Permanent fish cell lines can serve as a test system for the evaluation of a chemical's cytotoxicity. This paper presents research results on the cytotoxic effects on Channel Catfish Ovary (CCO) cell line induced by fifteen cholinium-based ionic liquids and deep eutectic solvents. Based on the decrease in cell viability, the most obvious toxic effect on CCO cells was caused by ionic liquid choline oxalate, while other solvents tested exhibited low cytotoxicity. Therefore, we can conclude that cholinium-based ionic liquids and deep eutectic solvents are comparatively less toxic to CCO cells than conventional ionic liquids., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fruit wines contain a wide range of phenolic compounds with biological effects, but their composition and potential benefits to human health have been studied to the much lesser extent compared to grape wines. The aim of this research is to study the phenolic profile of different types of fruit wines and to evaluate their antioxidant and biological potential. Commercially available fruit wines from blackberry, cherry, raspberry, blackcurrant, strawberry and apple produced in Croatia were analyzed. To the best of our knowledge, this study represents the first comprehensive screening of Croatian fruit wines. The phenolic characterization was performed by spectrophotometry and HPLC-PDA/MS analysis. The antioxidant capacity was determined using ABTS and FRAP assays, while in vitro biological activity was analyzed by the cytotoxicity assay on human breast (MCF-7), colon (CaCo-2) and cervical (HeLa) cancer cell lines. Among the studied fruit wines, blackberry, cherry and blackcurrant wines contained the highest amount of total phenolics, while the last two also contained the highest amount of total anthocyanins. The analysis of individual phenolic compounds showed distinctive phenolic composition of each type of fruit wine, notably as regards anthocyanins. Blackberry, followed by cherry, raspberry and blackcurrant wines also had a significantly higher antioxidant capacity than strawberry and apple wines. Fruit wines inhibited the growth of human cancer cells in vitro in a dose--dependent manner with differing susceptibility among tested cancer cells. Blackberry, cherry, raspberry and blackcurrant wines in the volume ratio of 10 and 20% showed to be the most effective anti-proliferative agents, with higher susceptibility in HeLa and MCF-7 cells than CaCo-2 cells.

In this work we describe the adaptation of channel catfish ovary (CCO) cell line to commercially available Ultra Culture serum-free medium by gradual reduction of serum concentration from 10 to 0 %. With this approach we obtained CCO cells fully adapted to serum-free conditions in 32 days. Growth, nutritional and morphological characteristics of these cells remained unchanged when compared to the control group kept in the presence of serum. Additionally, three commercially available protein hydrolysates were tested for the effects on growth performance of the newly serum-free adapted CCO cells. Supplementation with wheat gluten hydrolysate resulted in growth similar to serum free medium solely, while yeast and soy hydrolysates showed inhibitory effects on the cell growth. Taken together, the successful adaptation of CCO cells to serum-free conditions indicates their potential to be used in cytotoxicity assays when serum omission is demanded or for developing serum free bioprocesses using CCO cells. However, a more extended study on nutrient supplementation is still required to further boost the cell growth in a serum free culture.

Background: It remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. To achieve this, the understanding of the immunological parameters that mediate such broad protection is crucial., Method: In the current study we assessed the contribution of humoral and cellular immune responses to heterosubtypic protection against H5N1 induced by a Matrix-M (MM) adjuvanted seasonal influenza vaccine by serum transfer and T-cell depletion studies., Results: We demonstrate that the heterosubtypic protection against H5N1 induced by MM adjuvanted vaccine is partially mediated by antibodies. The serum contained both H5N1 cross-reactive hemagglutinin (HA)- and neuraminidase (NA)-specific antibodies but with limited virus neutralizing and no hemagglutination inhibiting activity. The cross-reactive antibodies induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting a role for the Fc part of the antibodies in protection against H5N1. Besides H5N1 specific antibody responses, cross-reactive HA- and NA-specific T-cell responses were induced by the adjuvanted vaccine. T-cell depletion experiments demonstrated that both CD4+ and CD8+ T cells contribute to protection., Conclusion: Our study demonstrates that cross-protection against H5N1 induced by MM adjuvanted seasonal virosomal influenza vaccine requires both the humoral and cellular arm of the immune system.

Background: Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal., Methods: To assess the immune potentiating properties of Matrix-M™, mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M™. Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M™ broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses., Results: Matrix-M™ adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M™ adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained., Conclusion: The results of this study emphasize the promising potential of a Matrix-M™-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunity.

Unlabelled: Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4 × 10(6) 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus., Importance: There is only very limited information on the effect of virus inoculation dose on the level of virus production and the induction of adaptive immune responses in humans or nonhuman primates. We found only a marginal and variable effect of virus dose on virus production in the trachea but a significant effect on body temperature. The induction of functional antibody responses, including virus neutralization titer, hemagglutination inhibition titer, and antibody-dependent cell-mediated cytotoxicity, correlated with the level of virus replication measured in the trachea. The study reveals a relationship between virus production and functional antibody formation, which could be relevant in defining appropriate criteria for new influenza virus vaccine candidates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

There is a constant threat of zoonotic influenza viruses causing a pandemic outbreak in humans. It is virtually impossible to predict which virus strain will cause the next pandemic and it takes a considerable amount of time before a safe and effective vaccine will be available once a pandemic occurs. In addition, development of pandemic vaccines is hampered by the generally poor immunogenicity of avian influenza viruses in humans. An effective pre-pandemic vaccine is therefore required as a first line of defense. Broadening of the protective efficacy of current seasonal vaccines by adding an adjuvant may be a way to provide such first line of defense. Here we evaluate whether a seasonal trivalent virosomal vaccine (TVV) adjuvated with the saponin-based adjuvant Matrix-M (MM) can confer protection against avian influenza H5 and H7 virus strains in mice and ferrets. We demonstrate that mice were protected from death against challenges with H5N1 and H7N7, but that the protection was not complete as evidenced by severe clinical signs. In ferrets, protection against H7N9 was not observed. In contrast, reduced upper and lower respiratory tract viral loads and reduced lung pathology, was achieved in H5N1 challenged ferrets. Together these results suggest that, at least to some extent, Matrix-M adjuvated seasonal virosomal influenza vaccine can serve as an interim measure to decrease morbidity and mortality associated with a pandemic outbreak.

Within this work we evaluated the cytotoxicity towards the Channel Catfish Ovary (CCO) cell line of some imidazolium-based ionic liquids containing different functionalized and unsaturated side chains. The toxic effects were measured by the reduction of the WST-1 dye after 72 h exposure resulting in dose- and structure-dependent toxicities. The obtained data on cytotoxic effects of 14 different imidazolium ionic liquids in CCO cells, expressed as EC50 values, were used in a preliminary quantitative structure-toxicity relationship (QSTR) study employing regression- and classification-based approaches. The toxicity of ILs towards CCO was chiefly related to the shape and hydrophobicity parameters of cations. A significant influence of the quantum topological molecular similarity descriptor ellipticity (ε) of the imine bond was also observed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Deep eutectic solvents (DESs) have been dramatically expanding in popularity as a new generation of environmentally friendly solvents with possible applications in various industrial fields, but their ecological footprint has not yet been thoroughly investigated. In the present study, three choline chloride-based DESs with glucose, glycerol and oxalic acid as hydrogen bond donors were evaluated for in vitro toxicity using fish and human cell line, phytotoxicity using wheat and biodegradability using wastewater microorganisms through closed bottle test. Obtained in vitro toxicity data on cell lines indicate that choline chloride: glucose and choline chloride:glycerol possess low cytotoxicity (EC50>10 mM for both cell lines) while choline chloride:oxalic acid possess moderate cytotoxicity (EC50 value 1.64 mM and 4.19 mM for fish and human cell line, respectively). Results on phytotoxicity imply that tested DESs are non-toxic with seed germination EC50 values higher than 5000 mg L(-1). All tested DESs were classified as'readily biodegradable' based on their high levels of mineralization (68-96%). These findings indicate that DESs have a green profile and a good prospect for a wider use in the field of green technologies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Our previous studies showed that alteration of dipeptides Y-Fca-Ala-OMe (III) into Y-Ala-Fca-OMe (IV) (Y=Ac, Boc; Fca=1'-aminoferrocene-1-carboxylic acid) significantly influenced their conformational space. The novel bioconjugates Y-Fca-Pro-OMe (1, Y=Ac; 2, Y=Boc) and Y-Pro-Fca-OMe (3, Y=Boc; 4, Y=Ac) have been prepared in order to investigate the influence of proline, a well-known turn-inducer, on the conformational properties of small organometallic peptides with an exchanged constituent amino acid sequences. For this purpose, peptides 1-4 were subjected to detailed spectroscopic analysis (IR, NMR, CD spectroscopy) in solution. The conformation of peptide 3 in the solid state was determined. Furthermore, the ability of the prepared conjugates to inhibit the growth of estrogen receptor-responsive MCF-7 mammary carcinoma cells and HeLa cervical carcinoma cells was tested.

Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing 'universal' protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain 'universal' protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines.

We studied the effects of five imidiazolium based ionic liquids with different anions and length of alkyl chains linked to imidazolium ring on the early development of barley (Hordeum vulgare). The inhibitory effect depends on the ionic liquids concentration and chemical structure, whereby the most toxic one was [C10mim][Br], followed by [C7mim][Br], [C4mim][Br], [C4mim][CH3CO2] and [C4mim][BF4]. Both anion and cation structures affected the toxicity of ionic liquid indicating that selection of more biocompatible anions such as [CH3CO2] does not necessarily indicate lower toxicity. Alternation in the extent of oxidative stress and antioxidant enzymes activities were found in barley plants due to ionic liquid treatments. When seedlings were exposed to higher concentrations of ionic liquids, antioxidant system could not effectively remove reactive oxidative species, leading to lipid peroxidation and damage of the photosynthetic system. However, overall data indicated that the performance of barley seedling was improved when all measured enzymes involved in scavenging of reactive oxygen species (ROS) were increased with special emphasis on GPX activities. Since there are no studies about ionic liquid (IL) toxicity in plants, that simultaneously evaluates the antioxidative enzyme system in response to different ILs, this work is valuable for gaining knowledge about the protection mechanism of plants from oxidative stress caused by IL exposure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Over past decades ionic liquids, a promising alternative to traditional organic solvents, have been dramatically expanding in popularity as a new generation of chemicals with potential uses in various areas in industry. In the literature these compounds have often been referred to as environmentally friendly; however, in recent years the perception of their greenness dramatically changed as the scientific community began to proactively assess the risk of their application based on the entire life-cycle. This review gives a brief overview of the current knowledge regarding the potential risks linked to the application of ionic liquids - from preparation to their disposal, with special emphasis on their potential environmental impacts and future directions in designing inherently safer ionic liquids., (© 2013 Published by Elsevier Inc.)

Increasing interest in the application of ionic liquids as green replacement for volatile organic solvents emphasized the need for the evaluation of their toxic effects at different biological systems in order to reduce the risk for human health and environment. To our knowledge, effects of imidazolium ionic liquids on cellular level of fish cell lines have not been studied yet. The cytotoxicity of imidazolium ionic liquids containing different anions and alkyl chain lengths as the substituent at the cation ring towards the fish CCO cell line was determined by WST-1 proliferation assay. Morphological alterations were examined by fluorescent microscopy using acridine orange/ethidium bromide staining and flow cytometry analysis was also performed. The results showed concentration-dependent cytotoxicity of ionic liquids in CCO cells, related to the type of anion and alkyl chain length, while EC50 values showed moderate to high cytotoxicity of tested imidazolium ionic liquids. Distinct morphological changes observed under fluorescence microscope and data obtained by flow cytometry suggest that the toxicity of imidazolium ionic liquids with longer alkyl chains could be related to necrosis. Results presented in here may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on aquatic environment., (Copyright © 2013 Elsevier Inc. All rights reserved.)