Recovering rhamnogalacturonan-I pectin from sugar beet pulp using a sequential ultrasound and microwave-assisted extraction: Study on extraction optimization and membrane purification.

This research focuses on the integrated recovery of rhamnogalacturonan-I (RG-I) pectin from sugar beet pulp (SBP). First, the extraction of RG-I pectin through sequential ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) was assessed. Optimization using a response surface methodology identified the optimal conditions as initial pH 4, 10 min of UAE, and 157 °C for MAE, achieving a 66.0 % recovery of pectooligosaccharides (POS). Additionally, purification through continuous diafiltration and concentration via ultrafiltration of the POS using membranes with different molecular weight cut-offs (MWCO) was explored. In contrast to previous research using discontinuous diafiltration, the use of continuous diafiltration allowed a decrease in the extract viscosity and obtained higher yields using a higher MWCO membrane. The refined RG-I pectin solids exhibited a high global yield (39-40 g pectin/100 g SBP), and high-methoxyl characteristics, as well as purity levels (70-80 %) similar to commercial prebiotics.

Efficient recovery and characterization of humic acids from municipal and manure composts: A comparative study.

The recovery of humic acids from low-quality compost obtained in municipal solid waste treatment plants provides opportunities for its valorization. This study compares the recovery and properties of the humic acids obtained from municipal mixed waste compost (MMWC) and manure compost. The effects of temperature, time, and KOH concentration on the ratio of humic acids in the extracted liquid and the content of organic carbon of the precipitates were investigated by response surface methodology. Optimal conditions were 30 °C and 24 h for both composts, with a KOH concentration of 0.53 M for MMWC and 0.25 M for manure compost. The manure compost provided a liquid extract richer in humic acids than MMWC (76.6 % vs. 33.7 %), but the precipitates presented similar organic carbon contents (38.1 % vs. 42.4 %). Regarding composition, both humic acids presented higher organic carbon and nitrogen contents than the composts used as feedstock. The extraction and further precipitation of humic acids reduced the concentration of heavy metals. Humic acids from manure compost have a slightly higher average molecular weight (2650 Da) than those from MMWC (1980 Da), while both present similar C/N ratios and degree of aromaticity. Most contaminants of emerging concern present in the original composts were not detected in the humic acids. Thus, it was demonstrated that MMWC constitutes an attractive source of humic acids with properties similar to those obtained from a high-quality compost and, therefore, with potential economic value.

Carrot Discard as a Promising Feedstock to Produce 2,3-Butanediol by Fermentation with P. polymyxa DSM 365.

The valorization of fruit and vegetable residues (such as carrot discard) and their microbial conversion into 2,3-butanediol (BDO) can be considered as a very interesting way to reduce food waste and sustainably originate high value-added products. This work analyzes the valorization of carrot discard as feedstock for 2,3-butanediol (BDO) production by Paenibacillus polymyxa DSM 365. The influences of stirring and the presence of tryptone (nitrogen source) are studied. Furthermore, in order to evaluate the influence of the pre-culture medium (nitrogen source, nutrients, and pH) and the substrate, fermentation assays in simple and mixture semi-defined media (glucose, fructose, and/or galactose) were also carried out. As a result, 18.8 g/L BDO, with a BDO yield of 0.43 g/g (86% of its theoretical value), could be obtained from carrot discard enzymatic hydrolysate at 100 rpm, no tryptone, and pre-culture Häßler medium. No hydrothermal pre-treatment was necessary for BDO production from carrot discard, which increases the profitability of the process. Therefore, 18.8 g BDO, as well as 2.5 g ethanol and 2.1 g acetoin by-products, could be obtained from 100 g of carrot discard (dry matter).

Synthesis, Biological Evaluation, and Docking Studies of Open-Chain Carbohydrate Amides as Acetylcholinesterase Inhibitors.

INTRODUCTION: Alzheimer's disease is a multifactorial syndrome, which is not yet fully understood, causing memory loss, dementia, and, ultimately, death. Acetylcholinesterase inhibitors are the mainstay drugs that are used in disease-symptomatic treatment. In this work, we report a new synthetic route yielding sugar amides as low to moderate acetylcholinesterase inhibitors. METHODS: Commercially available diacetone glucose was converted into perbenzyl D-glucono-1,4- lactone, which reacted with aromatic or aliphatic amines to afford the corresponding new amides in a high isolated yield. Docking studies of the most promising hydroxybutylamide and benzylamide were performed to assign binding interactions with acetylcholinesterase and determine the key features for bioactivity. RESULTS: The inhibitors are accommodated in enzyme gorge, blocking the access to Ser203 mainly due to π-π stacking interactions of sugar benzyl groups with the aromatic gorge residues, Tyr337 and Tyr341 for both inhibitors and Trp439 only for the hydroxybutylamide. CONCLUSION: Bonding is also significant through sugar interaction with the residues Tyr124 and Ser125-OH in both inhibitors. Flexibility of these open-chain structures seems to be quite relevant for the observed binding to acetylcholinesterase.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Liquid fertilizer production from organic waste by conventional and microwave-assisted extraction technologies: Techno-economic and environmental assessment.

The use of mineral fertilizers in agriculture has significantly increased to support the growing global food demand. Organic fertilizers are produced from renewable waste materials to overcome the drawbacks of inorganic fertilizers. The development of novel production processes of organic fertilizers entails a significant advance towards the circular economy that reincorporates waste materials into the production cycle. In this work, the economic and environmental feasibility of an industrial plant with a treatment capacity of 300 kg/h of organic waste for the production of liquid fertilizers has been performed. Two extraction technologies (conventional and microwave) and two solvents (water and alkaline) have been compared to select the most sustainable and profitable scenario for scaling-up. The extraction process consists of 2 steps: extraction followed by a concentration stage (necessary only if water extraction is applied). The resolution of the mass balances shows that the fertilizer production under alkaline conditions is ten times higher than for water-based extraction. The economic analysis demonstrated that the total investment cost of microwave technology (>3.5 M€) is three times higher compared to the conventional extraction technology (<1.5 M€), mainly due to the higher complexity of the equipment. These facts directly impact the minimum selling price, because the fertilizers obtained by conventional extraction with alkaline solvent would have a lower selling price (about 1 €/L). As for environmental assessment, the indicators show that the environmental impact produced by water-based extraction is higher than alkaline-solvent extraction, mainly due to the necessity of a concentration stage of the liquid extract to meet the requirements of European regulations. In view of the results obtained in the economic and environmental evaluation, it could be concluded that the most favourable scenario for scaling up the production of liquid fertilizers from organic waste is the conventional extraction under alkaline conditions.

Ideal conditions of microwave-assisted acid pretreatment of sugarcane straw allow fermentative butyric acid production without detoxification step.

Sugarcane straw (SCS) was pretreated with dilute sulfuric acid assisted by microwave to magnify fermentable sugars and to minimize the concentration of inhibitors in the hydrolysates. The optimum conditions for maximum recovery of sugars were 162 °C and 0.6% (w/v) H2SO4. The low level of inhibitors, such as acetate (2.9 g/L) and total phenolics (1.4 g/L), in the SCS slurry from the pretreatment stage allowed the enzymatic hydrolysis and fermentation steps to occur without detoxification. Besides consuming the total sugar content (31.0 g/L), Clostridium beijerinckii Br21 was able to use acetate from the SCS hydrolysate, to give butyric acid at high conversion factor (0.49 g of butyric acid /g of sugar). The optimized pretreatment conditions spared acid, time, and the detoxification stage, making bio-butyric acid production from SCS extremely attractive.

Efficient biobutanol production by acetone-butanol-ethanol fermentation from spent coffee grounds with microwave assisted dilute sulfuric acid pretreatment.

The integral valorization of potential sugars (cellulosic and hemicellulosic) from spent coffee grounds (SCG), a lignocellulosic residue, is proposed in this work. With this aim, the microwave assisted dilute sulfuric acid pretreatment has been optimized, leading to a hemicellulosic sugar recovery in the pretreatment liquid (HSRL) and an enzymatic hydrolysis yield of 79 and 98%, respectively, at 160.47 °C and 1.5% H2SO4. Moreover, the complete digestibility of cellulose (enzymatic hydrolysis yield = 100%) was also discovered for non-pretreated SCG, which is very interesting. Secondly, the production of biobutanol, an advanced biofuel, is also proposed from pretreated SCG enzymatic hydrolysate and pretreatment liquid achieved under optimal conditions. These were fermented by Clostridium beijerinckii, yielding 95 kg butanol/t SCG (dry matter) and 151 kg acetone-butanol-ethanol/t SCG (dry matter).

3-Oxo-ß-sultam as a Sulfonylating Chemotype for Inhibition of Serine Hydrolases and Activity-Based Protein Profiling.

3-Oxo-ß-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-ß-sultam inhibitors and show, through crystallographic data, that they are regioselective sulfonylating electrophiles, covalently binding to the catalytic serine of human and porcine elastases through the sulfur atom. Application of 3-oxo-ß-sultam-derived activity-based probes in a human proteome revealed their potential to label disease-related serine hydrolases and proteasome subunits. Activity-based protein profiling applications of 3-oxo-ß-sultams should open up new opportunities to investigate these classes of enzymes in complex proteomes and expand the toolbox of available sulfur-based covalent protein modifiers in chemical biology.

Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential.

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer's disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman's assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of Ki = 4.29 µM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing Ki = 1.72 µM and Ki' = 1.28 µM, respectively.

A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

Biobutanol production from brewer's spent grain hydrolysates by Clostridium beijerinckii.

Brewer's spent grain (BSG) is a promising feedstock for ABE fermentation. Sulfuric acid pretreatment of BSG at pH 1, 121°C and different solid loadings (5-15% w/w) was investigated. Enzymatic hydrolysis and ABE fermentation by Clostridium beijerinckii DSM 6422 of non-washed and washed pretreated BSG were performed to compare monosaccharide release and butanol production. Pretreatment at 15% w/w BSG resulted in higher availability of sugars in both the enzymatic hydrolysates and pretreatment liquid, and overall yields of 75gbutanol/kg BSG and 95gABE/kg BSG were obtained. When the enzymatic hydrolysate from the washed pretreated BSG was fermented, butanol (6.0±0.5g/L) and ABE (7.4±1.0g/L) concentrations were lower compared with 7.5±0.6g/L butanol and 10.0±0.8g/L ABE from a control. The fermentation of the liquid released in the pretreatment at 15% w/w resulted in a butanol production of 6.6±0.8g/L with a total ABE of 8.6±1.3g/L after overliming.

Dipeptidyl Vinyl Sulfone as a Novel Chemical Tool to Inhibit HMGB1/NLRP3-Inflammasome and Inflamma-miRs in Aß-Mediated Microglial Inflammation.

Rapid microglial activation and associated inflammatory pathways contribute to immune-defense and tissue repair in the central nervous system (CNS). However, persistent activation of these cells will ultimately result in vast production of pro-inflammatory mediators and other neurotoxic factors, which may induce neuronal damage and contribute to chronic neurodegenerative diseases, as Alzheimer's disease (AD). Therefore, small molecules with immunomodulatory effects on microglia may be considered as potential tools to counteract their proinflammatory phenotype and neuroimmune dysregulation in such disorders. Indeed, reducing amyloid-ß (Aß)-induced microglia activation is believed to be effective in treating AD. In this study, we investigated whether dipeptidyl vinyl sulfone (VS) was able to attenuate Aß-mediated inflammatory response using a mouse microglial (N9) cell line and a solution containing a mixture of Aß aggregates. We show that low levels of VS are able to prevent cell death while reducing microglia phagocytosis upon Aß treatment. VS also suppressed Aß-induced expression of inflammatory mediators in microglia, such as matrix metalloproteinase (MMP)-2 and MMP-9, as well as high-mobility group box protein-1 (HMGB1), nod-like receptor protein 3 (NLRP3)-inflammasome, and interleukin (IL)-1ß. Interestingly, increased expression of the two critical inflammation-related microRNAs (miR)-155 and miR-146a in microglia upon Aß treatment was also prevented by VS coincubation. Taken together, VS emerges as a potential new therapeutic strategy worthy of further investigation in improved cellular and animal models of AD.

Clickable 4-Oxo-ß-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes.

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC, or 'click' chemistry) approach based on the 4-oxo-ß-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure-activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-ß-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC50 values in the nanomolar range (56-118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools.

hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus.

Wittig Reaction: Domino Olefination and Stereoselectivity DFT Study. Synthesis of the Miharamycins' Bicyclic Sugar Moiety.

2-O-Acyl protected-d-ribo-3-uloses reacted with [(ethoxycarbonyl)methylene]triphenylphosphorane in acetonitrile to afford regio- and stereoselectively 2-(Z)-alkenes in 10-60 min under microwave irradiation. This domino reaction is proposed to proceed via tautomerization of 3-ulose to enol, acyl migration, tautomerization to the 3-O-acyl-2-ulose, and Wittig reaction. Alternatively, in chloroform, regioselective 3-olefination of 2-O-pivaloyl-3-uloses gave (E)-alkenes, key precursors for the miharamycins' bicyclic sugar moiety.

Converting maslinic acid into an effective inhibitor of acylcholinesterases.

During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related derivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholinesterases, up to now maslinic acid has not been part of such studies. For this reason, three series of maslinic acid derivatives possessing modifications at different centers were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants comparable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore, additional selectivity as well as cytotoxicity studies were performed underlining the potential of several derivatives and qualifying them for further investigations. Docking studies revealed that the different kinetic behavior within the same compound series may be explained by the ability of the compounds to enter the active site gorge of AChE.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties.

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

Efficient acetone-butanol-ethanol production by Clostridium beijerinckii from sugar beet pulp.

Sugar beet pulp (SBP) has been investigated as a promising feedstock for ABE fermentation by Clostridium beijerinckii. Although lignin content in SBP is low, a pretreatment is needed to enhance enzymatic hydrolysis and fermentation yields. Autohydrolysis at pH 4 has been selected as the best pretreatment for SBP in terms of sugars release and acetone and butanol production. The best overall sugars release yields from raw SBP ranged from 66.2% to 70.6% for this pretreatment. The highest ABE yield achieved was 0.4g/g (5.1g/L of acetone and 6.6g/L butanol) and 143.2g ABE/kg SBP (62.3g acetone and 80.9g butanol) were obtained when pretreated SBP was enzymatically hydrolyzed at 7.5% (w/w) solid loading. Higher solid loadings (10%) offered higher acetone and butanol titers (5.8g/L of acetone and 7.8g/L butanol). All the experiments were carried out under not-controlling pH conditions reaching about 5.3 in the final samples.

Human Staufen1 associates to miRNAs involved in neuronal cell differentiation and is required for correct dendritic formation.

Double-stranded RNA-binding proteins are key elements in the intracellular localization of mRNA and its local translation. Staufen is a double-stranded RNA binding protein involved in the localised translation of specific mRNAs during Drosophila early development and neuronal cell fate. The human homologue Staufen1 forms RNA-containing complexes that include proteins involved in translation and motor proteins to allow their movement within the cell, but the mechanism underlying translation repression in these complexes is poorly understood. Here we show that human Staufen1-containing complexes contain essential elements of the gene silencing apparatus, like Ago1-3 proteins, and we describe a set of miRNAs specifically associated to complexes containing human Staufen1. Among these, miR-124 stands out as particularly relevant because it appears enriched in human Staufen1 complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro. In agreement with these findings, we show that expression of human Staufen1 is essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state, and suggest potential human Staufen1 mRNA targets involved in this process.