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Abstract Tissue paper was the only paper grade whose consumption increased during 2020 in Europe. In a highly competitive context, this work explores a strategy based on bisacrylamide cross-linkers and poly(vinyl alcohol) (PVA), seeking to enhance the water uptake of pulps for tissue paper and the key properties of the resulting tissue sheets: water absorption capacity, capillarity, softness, porosity, and strength. For that, α-cellulose from cotton and a kraft hardwood pulp, in parallel, were reacted with N,N’-methylenebisacrylamide, both in the absence and in the presence of PVA. The water desorption rate of the modified polymers was monitored. Pulp blends were then mixed with a conventional softwood pulp (30%) to prepare laboratory tissue paper sheets (20 g m–2). For cotton cellulose, cross-linking with PVA more than doubled the water uptake, up to 7.3 g/g. A significant enhancement was also obtained in the case of pulps, up to 9.6 g/g, and in the case of paper, to 11.9 g/g. This improvement was consistent with a drastic increase in porosity, and it was not detrimental to paper strength. Graphical Abstract

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI–MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2–8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80–90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319–541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs. Graphical abstract: [Figure not available: see fulltext.]

N-containing heterocycles (NCHs) are largely used as precursors for pharmaceuticals and can enter the environment. Some NCHs have been shown to be toxic, persistent, and very mobile in the environment. Thus, they have received increasing attention in the past years. However, the analysis of these polar compounds in environmental samples is still a challenge for liquid chromatography. This paper investigates the use of mixed-mode liquid chromatography (MMLC), which has reversed-phase and ion exchange characteristics for measurements of NCHs in water. NCHs with low pKa (i.e., Ka (i.e., > 5) interact by a mixture of reversed-phase/ion exchange/HILIC mechanism. It was also shown that the presented method performs well in the quantification of the majority of the selected NCHs in surface water with MDLs between 3 and 6 μg/L, a low matrix effect and recoveries in the range of 77–96% except for pyridazine exhibiting 32% were achieved. The method was successfully employed to follow the degradation of NCHs in ozonation.

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 μg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC R2 0.86–0.98 and 0.86–0.98 linearity, relative standard deviation QC Graphical abstract

new strategy for the computer-assisted methods development in the reversed-phase liquid chromatographic separations of unknown sample mixtures has been developed using the latent spectral information in chromatogram raw data files of appropriately designed experiments, rather than resorting to elemental information functions (e.g., the number of peaks in chromatograms or similar criteria). The strategy developed allows unification of the approach for samples of both known and unknown composition and, thus, provide a general strategy for computer-aided tools in the chromatography laboratory. The operation principle of this strategy departs from extracting the spectra of components in the mixture chromatograms by resorting to multivariate curve resolution-alternating least squares (MCR-ALS). This technique allows the estimation of the true spectra for the individual components except when they have identical spectra or are fully overlapped. Thus, a convenient experimental design will try to perform separations of the sample mixture having at least partial resolution of components in some runs. This will allow estimating the spectra of components and, then, assign these components to the peaks in each run chromatogram. In this way, a retention model can be built for each component so computerized optimization process can be developed to provide the chromatographer with the best possible separation programs. Following this approach, strategies for sample mixtures of known and unknown composition are only different in the need of an initial spectrum discovery process for unknown mixtures and therefore a real general approach for the computer-assisted LC methods development is now available for the first time Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by GAIN (Axencia Galega de Innovación) through the Programa Industrias de Futuro 4.0 and co-financed by the European Regional Development Fund (ERDF) under the Feder Galicia 2014-2020 operational program and the Consellería de Economía, Emprego e Industria within the project “Laboratorio Dixital do Futuro (FutureLab” (IN854A 2019/16) SI

Analytical and bioanalytical chemistry 412(6), 1263-1275 (2020). doi:10.1007/s00216-019-02296-x special issue: "Detection of E. coli using lateral flow assay and LIBS/Embedding carbon dots and gold nanoclusters in MOFs/Sorbent-incorporated dipstick for direct assaying of proteases/Mammalian ovarian lipid distributions by DESI-MS imaging", Published by Springer, Berlin ; Heidelberg

Recent years have confirmed the ubiquitous applicability of lateral flow assays (LFA) in point-of-care testing (POCT). To make this technology available for low abundance analytes, strategies towards lower limits of detections (LOD), while maintaining the LFA’s ease of use, are still being sought. Here, we demonstrate how liposomes can significantly improve the LOD of traditional gold nanoparticle (AuNP)–based assays while fully supporting a ready-to-use system for commercial application. We fine-tuned liposomes towards photometric and fluorescence performance on the synthesis level and applied them in an established interleukin 6 (IL-6) immunoassay normally using commercial AuNP labels. IL-6’s low abundance (−1) and increasing relevance as prognostic marker for infections make it an ideal model analyte. It was found that liposomes with a high encapsulant load (150 mmol L−1 sulforhodamine B (SRB)) easily outperform AuNPs in photometric LFAs. Specifically, liposomes with 350 nm in diameter yield a lower LOD even in complex matrices such as human serum below the clinically relevant range (7 pg mL−1) beating AuNP by over an order of magnitude (81 pg mL−1). When dehydrated on the strip, liposomes maintained their signal performance for over a year even when stored at ambient temperature and indicate extraordinary stability of up to 8 years when stored as liquid. Whereas no LOD improvement was obtained by exploiting the liposomes’ fluorescence, an extraordinary gain in signal intensity was achieved upon lysis which is a promising feature for high-resolution and low-cost detection devices. Minimizing the procedural steps by inherently fluorescent liposomes, however, is not feasible. Finally, liposomes are ready for commercial applications as they are easy to mass-produce and can simply be substituted for the ubiquitously used AuNPs in the POCT market. Graphical abstract

[EN]The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to G(i/o) proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 +/- 10.5 nm at pH 3 (25 oC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 +/- 15.2 nm at 35 degrees C. Lower critical solution temperature of this polymer was found to be approximate to 33.4 degrees C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1(414-472) and GST-CB1(414-442) were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR). Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for this research was provided by the Spanish Ministry of Science, Innovation and Universities (project CTQ2017-85686-R) and by the Basque Government (Research Groups of the Basque University System, Project No IT 1186-19.

The extensive use of silver nanoparticles (AgNPs) in consumer products, medicine, and industry leads to their release into the environment. Thus, a characterization of the concentration, size, fate, and toxicity of AgNPs under environmental conditions is required. In this study, we present the characterization and optimization of an asymmetric flow field-flow fractionation (AF4) system coupled with UV/Vis spectrophotometer and dynamic light scattering (DLS) detector as a powerful tool for the size separation and multi-parameter characterization of AgNPs in complex matrices. The hyphenated AF4-UV/Vis-DLS system was first characterized using individual injections of the different size fractions. We used electrostatically stabilized AgNPs of 20-, 50-, and 80-nm nominal diameters coated with lipoic acid. We investigated the effect of applied cross-flows, carrier solutions, focus times, and quantity of injected particles on the nature of the AF4 fractograms and on the integrity of the AgNPs. Best size separation of a 1:1 mixture of 20- and 80-nm AgNPs was achieved using cross-flows of 0.5 and 0.7 mL/min with 1 mM NaCl and 0.05% v/v Mucasol as carrier solutions. We also researched the behavior of AgNPs in natural waters using the hyphenated AF4-UV/Vis-DLS system, under determined optimal conditions. Open Access funding enabled and organized by Projekt DEAL. This research was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (grant agreement number PIEF-GA-2012-329575) and the EU research project “ExPloTect-Ex-situ, near-real-time explosive compound detection in sea- water”co-funded by the EMFF programme of the European Union [grant agreement number 863693]. P.L. is supported by a Beatriz Galindo Senior Fellowship (BG20/00104) from Ministerio de Ciencia, Innovación y Universidades of Spain, and University of Lleida. F.G. acknowledges the German Federal Ministry of Economic Affairs and Energy [OCEANSensor project; Funding Agreement 03SX459A] for funding.

In this study, a highly sensitive, electrochemical, and label-free DNA impedimetric sensor was developed using carbonized glass fiber–coal tar pitch (GF–CTP) electrodes supported with gold nanoparticles (AuNPs) for the detection of HIV-1 gene. Thiol-modified GF–CTP electrodes were prepared using amine crosslinking chemistry and AuNPs were self-assembled obtaining highly conductive nanoelectrodes, GF–CTP–ATP–Au. All steps of electrode modifications were characterized using electrochemical, spectroscopic, and microscopic methods. GF–CTP–ATP–Au electrode was then modified with a capture DNA probe (C-ssDNA) and optimized with a target DNA probe in terms of hybridization time and temperature between 30 and 180 min and 20–50 °C, respectively. Finally, the analytic performance of the developed ssDNA biosensor was evaluated using electrochemical impedance spectroscopy. The calibration of the sensor was obtained between 0.1 pM and 10 nM analyte working range. The limit of detection was calculated using signal to noise ratio of 3 (S/N = 3) as 13 fM. Moreover, interference results for two noncomplementary DNA probes were also tested to demonstrate non-specific ssDNA interactions. An electrochemical label-free DNA impedimetric sensor was successfully developed using a novel GF–CTP–ATP–Au electrode. This study suggests that highly sensitive DNA-based biosensors can be developed using relatively low-cost carbonaceous materials. © 2020, Institute of Chemistry, Slovak Academy of Sciences.

Small heat shock proteins (sHSPs) are found in all three domains of life (Bacteria, Archaea, and Eukarya) and play a critical role in protecting organisms from a range of environmental stresses. However, little is known about their physiological functions in red algae. Therefore, we characterized the sHSPs (PysHSPs) in the red macroalga Pyropia yezoensis, which inhabits the upper intertidal zone where it experiences fluctuating stressful environmental conditions on a daily and seasonal basis, and examined their expression profiles at different developmental stages and under varying environmental conditions. We identified five PysHSPs (PysHSP18.8, 19.1, 19.2, 19.5, and 25.8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that expression of the genes PysHSP18.8, PysHSP19.5, and PysHSP25.8 was repressed at all the developmental stages under normal conditions, whereas PysHSP19.1 and PysHSP19.2 were overexpressed in mature gametophytes and sporophytes. Exposure of the gametophytes to high temperature, oxidative stress, or copper significantly increased the mRNA transcript levels of all the five genes, while exogenous application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) significantly increased the expression levels of PysHSP19.2, PysHSP19.5, and PysHSP25.8. These findings will help to further our understanding of the role of PysHSP genes and provide clues about how Pyropia species can adapt to the stressful conditions encountered in the upper intertidal zone during their life cycle. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-018-00959-9) contains supplementary material, which is available to authorized users.

Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-018-0930-1) contains supplementary material, which is available to authorized users.

Electrochemical immunosensors enable rapid analyte quantification in small sample volumes, and have been demonstrated to provide high sensitivity and selectivity, simple miniaturization, and easy sensor production strategies. As a point-of-care (POC) format, user-friendliness is equally important and most often not combinable with high sensitivity. As such, we demonstrate here that a sequence of metal oxidation and reduction, followed by stripping via differential pulse voltammetry (DPV), provides lowest limits of detection within a 2-min automatic measurement. In exchanging gold nanoparticles (AuNPs), which dominate in the development of POC sensors, with silver nanoparticles (AgNPs), not only better sensitivity was obtained, but more importantly, the assay protocol could be simplified to match POC requirements. Specifically, we studied both nanoparticles as reporter labels in a sandwich immunoassay with the blood protein biomarker NT-proBNP. For both kinds of nanoparticles, the dose-response curves easily covered the ng∙mL−1 range. The mean standard deviation of all measurements of 17% (n ≥ 4) and a limit of detection of 26 ng∙mL−1 were achieved using AuNPs, but their detection requires addition of HCl, which is impossible in a POC format. In contrast, since AgNPs are electrochemically less stable, they enabled a simplified assay protocol and provided even lower LODs of 4.0 ng∙mL−1 in buffer and 4.7 ng∙mL−1 in human serum while maintaining the same or even better assay reliability, storage stability, and easy antibody immobilization protocols. Thus, in direct comparison, AgNPs clearly outperform AuNPs in desirable POC electrochemical assays and should gain much more attention in the future development of such biosensors.

Analytical and bioanalytical chemistry (2021). doi:10.1007/s00216-021-03798-3, Published by Springer, Berlin ; Heidelberg

Graphic abstract Nutritional information of burgers launched in the EU market during 2020 was retrieved from their labels. Products were initially classified into four types: i.e., veggie, red meat-, fish- and poultry-based. Gluten, wheat, and soy were the most declared allergens regardless of the burgers type. Veggie burgers showed levels of energy, fat, and saturate fatty acids (SFA) similar to fish- and poultry-based burgers, but lower than red meat burgers. Compared to conventional burgers, veggie had higher amounts of carbohydrates and sugars and a lower content of proteins, but no difference in salt. Due to the high compositional intra-variability in the veggie burgers, vegetarian and vegan burgers were further analyzed separately. Vegan burgers had higher levels of energy, fat, SFA and protein than vegetarian burgers, but lower carbohydrates and sugars. Once again large differences in the nutritional parameters were observed in both vegan and vegetarian burger categories due to the wide range of ingredients and formulations employed in these types of products. A clear and comprehensive informative labeling is especially needed for vegan and vegetarian burgers to allow the consumer to make a rational decision based on the nutritional facts of these products. Supplementary Information The online version contains supplementary material available at 10.1007/s00217-021-03808-9.

New psychoactive substances (NPS) have been introduced into the market in recent years, with new analytes reported every year. The use of these substances in women can occur at any stage of life, even in the childbearing age. Drug use during pregnancy presents significant risks for the mother and the fetus, so it is important to have tools that allow to detect prenatal exposure to these substances of abuse. Therefore, an analytical method for the determination of 137 NPS and other drugs of abuse in meconium by UHPLC-QTOF was developed and validated for semi-quantitative purpose. Linearity range, limit of detection (LOD), precision, matrix effect, selectivity, and specificity were evaluated. For all analytes, the calibration curves were studied in the ranges between 2, 10, or 50 ng/g and 750 or 1000 ng/g, (depending on the analyte) and the LOD ranged between 0.04 and 2.4 ng/g. The method was applied to 30 meconium specimens from cases in which fentanyl had been administered as epidural anesthesia at the time of delivery or cases in which the maternal hair was positive to other drug of abuse. Four meconium samples tested positive for fentanyl (range concentration = 440–750 ng/g) and two samples tested positive to acetylfentanyl (range concentration = 190–1400 ng/g) A. López-Rabuñal would like to thank the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, for her predoctoral contract (ED481A-2019/071) SI

The performance of two different analytical methodologies to investigate the presence of glyphosate (GLY) and aminomethylphosphonic acid (AMPA) residues in wine samples was evaluated. Transformation of compounds in their fluorene-9-methyloxycarbonyl derivatives permitted their separation under reversed-phase liquid chromatography with tandem mass spectrometry (LC–MS/MS) determination. Although the wine matrix severely impaired the efficiency of GLY derivatization, this drawback was solved using a molecularly imprinted sorbent for the previous, selective extraction of GLY and AMPA from wine. Alternatively, the use of a strong anionic exchange, polyvinyl alcohol-based LC column, turned to be the most effective alternative for direct determination of both compounds in diluted wine samples. The chromatographic behavior of this column and the magnitude of matrix effects observed during analysis of diluted wine samples were significantly affected by the composition of the mobile phase. Under final working conditions, this column permitted the separation of AMPA and the fungicide fosetyl (which shows common transitions in tandem MS/MS methods), it improved significantly the sample throughput versus extraction-derivatization-purification method, and it allowed the use of solvent-based calibration standards. Both analytical procedures provided similar limits of quantification (LOQs) for GLY (0.5–1.0 ng mL−1), while the multistep method was 8 times more sensitive to AMPA than the direct procedure. GLY residues stayed above method LOQs in 70% of the processed wines; however, concentrations measured in 95% of positive samples remained 100 times below the maximum residue limit (MRL) set for GLY in vinification grapes Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was supported by Spanish Government and Xunta de Galicia through grants PGC2018-094613-B-I00 and ED431C 2021/06, respectively. Both projects are co-funded by the EU FEDER program SI

Analytical and bioanalytical chemistry 413(12), 3253-3268 (2021). doi:10.1007/s00216-021-03261-3 special issue: "Acylation as derivatization strategy during an OPCW Proficiency Test/Comprehensive prognosis risk prediction of hepatocellular carcinoma/3D printed microdevice for smartphone-based on-site-capable uranium analysis/Intermolecular FRET sensor for tracking glycosylation of CD147 in living cells", Published by Springer, Berlin ; Heidelberg

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions. Electronic supplementary material The online version of this article (10.1007/s00203-020-01864-5) contains supplementary material, which is available to authorized users.

Multi-block glucans comprising permethylated and partially methylated blocks are compounds of interest. In order to monitor their formation by transglycosylation of corresponding starting glucans, a method has been developed and applied to model compounds. This method allows determining the average length of the blocks and the progress of incorporation of methyl blocks in partially methylated sequences with a random distribution. The method, comprising liquid chromatography mass spectrometry (LC-MS) and electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MSn) measurements of two types of peralkylated glucans representing derivatives of the target compounds, is comprehensively described and discussed. ESI-MSn allows looking into the sequences of oligomeric domains. In addition, transglycosylation is followed by attenuated total reflection FTIR and NMR spectroscopy. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02388-z) contains supplementary material, which is available to authorized users.

KURT KIZILDOGAN, ASLIHAN/0000-0002-9323-0993 WOS: 000530831200012 PubMed: 31833007 lysA gene encoding meso-diaminopimelic acid (DAP) decarboxylase enzyme that catalyzes l-lysine biosynthesis in the aspartate pathway in Streptomyces clavuligerus was overexpressed, and its effects on cephamycin C (CephC), clavulanic acid (CA), and tunicamycin productions were investigated. Multicopy expression of lysA gene under the control of glpF promoter (glpFp) in S. clavuligerus pCOlysA led to higher expression levels ranging from 2- to 6-fold increase at both lysA gene and CephC biosynthetic gene cluster at T-36 and T-48 of TSBG fermentation. These results accorded well with CephC production. Thus, 1.86- and 3.14-fold higher volumetric as well as 1.26- and 1.71-fold increased specific CephC yields were recorded in S. clavuligerus pCOlysA in comparison with the wild-type and its control strain, respectively, at 48th h. Increasing the expression of lysA provided 4.3 times more tunicamycin yields in the recombinant strain. These findings suggested that lysA overexpression in S. clavuligerus made the strain more productive for CephC and tunicamycin. The results also supported the presence of complex interactions among antibiotic biosynthesis pathways in S. clavuligerus. Ondokuz Mayis UniversitesiOndokuz Mayis University [PYO.ZRT.1902-A.15.001]

Analytical and bioanalytical chemistry 412(23), 5601-5613 (2020). doi:10.1007/s00216-020-02776-5 special issue: "NIR excitation/emission fluorescent probe for imaging of cysteine/SCF-MS for analyzing chemicals of emerging concern/CE/MC-ICP-MS for on-line species-specific isotopic analysis of sulfur/Novel alkaline phosphatase activity sensing strategy", Published by Springer, Berlin

Baobab is an endemic African plant rich in health-promoting compounds and still not extensively studied. Characterization of bioactive molecules in baobab fruit by means of LC system coupled with a photodiode array and a triple quadrupole mass spectrometer detector is here reported. This work provides a qualitative and quantitative profile of polyphenols in baobab pulp, shell, kernel and seeds. Baobab pulp is a rich source of ascorbic acid, (−)-epicatechin and procyanidin B2 (135.6, 104.8, and 100.1 mg/100 g). Moreover, a HPLC preparative method to isolate procyanidin fraction from pulp was developed, revealing how the antioxidant activity of baobab pulp not only depend on the ascorbic acid content but also on the presence of procyanidins. The use of baobab fruit pulp as promising healthy food or food supplement is also discussed.

Putative iron-reductase (IR) genes from Serpula lacrymans with similarity to the conserved iron-binding domains of cellobiose dehydrogenase (CDH) enzymes have been identified. These genes were cloned and expressed to functionally characterize their activity and role in the decomposition of lignocellulose. The results show that IR1 and IR2 recombinant enzymes have the ability to depolymerize both lignin and cellulose, are capable of the reduction of ferric iron to the ferrous form, and are capable of the degradation of nitrated lignin. Expression of these genes during wheat straw solid-state fermentation was shown to correlate with the release of compounds associated with lignin decomposition. The results suggest that both IR enzymes mediate a non-enzymatic depolymerisation of lignocellulose and highlight the potential of chelator-mediated Fenton systems in the industrial pre-treatment of biomass. Electronic supplementary material The online version of this article (10.1007/s10295-019-02238-7) contains supplementary material, which is available to authorized users.

Meningiomas are the commonest types of tumours in the central nervous system (CNS). It is a benign type of tumour divided into three WHO grades (I, II and III) associated with tumour growth rate and likelihood of recurrence, where surgical outcomes and patient treatments are dependent on the meningioma grade and histological subtype. The development of alternative approaches based on attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy could aid meningioma grade determination and its biospectrochemical profiling in an automated fashion. Herein, ATR-FTIR in combination with chemometric techniques is employed to distinguish grade I, grade II and grade I meningiomas that re-occurred. Ninety-nine patients were investigated in this study where their formalin-fixed paraffin-embedded (FFPE) brain tissue samples were analysed by ATR-FTIR spectroscopy. Subsequent classification was performed via principal component analysis plus linear discriminant analysis (PCA-LDA) and partial least squares plus discriminant analysis (PLS-DA). PLS-DA gave the best results where grade I and grade II meningiomas were discriminated with 79% accuracy, 80% sensitivity and 73% specificity, while grade I versus grade I recurrence and grade II versus grade I recurrence were discriminated with 94% accuracy (94% sensitivity and specificity) and 97% accuracy (97% sensitivity and 100% specificity), respectively. Several wavenumbers were identified as possible biomarkers towards tumour differentiation. The majority of these were associated with lipids, protein, DNA/RNA and carbohydrate alterations. These findings demonstrate the potential of ATR-FTIR spectroscopy towards meningioma grade discrimination as a fast, low-cost, non-destructive and sensitive tool for clinical settings. Graphical abstractAttenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to discriminate meningioma WHO grade I, grade II and grade I recurrence tumours. Electronic supplementary material The online version of this article (10.1007/s00216-019-02332-w) contains supplementary material, which is available to authorized users.

Dissolving grade pulps are used to manufacture regenerated cellulosic fibres. One promising process for the production of regenerated fibres utilises endoglucanse rich cellulases in the modification of dissolving pulp into alkaline soluble form. The aim of this paper was to characterise cellulases produced by Trichoderma reesei that are available in large quantities and study their effect on the dissolving grade softwood pulp, especially on its alkaline solubility. All the studied cellulases had endoglucanse activity and they decreased the intrinsic viscosity of the pulp. The degradation of cellulose into solubilised sugars increased with the cellulases containing also cellobiohydrolases. The monocomponent endoglucanases enhanced alkaline solubility of the pulp more than the multicomponent cellulases and produced alkaline solutions with higher fluidity. The studies showed that the type of the cellulases in the enzyme mixture has significant effect on the amount of solubilised sugars during the enzyme treatment and on the alkaline solubility of the pulp.

Aluminium is biologically reactive and its ability to potentiate the immune response has driven its inclusion in both veterinary and human vaccines. Consequently, the need for unequivocal visualisation of aluminium in vivo has created a focused research effort to establish fluorescent molecular probes for this purpose. The most commonly used direct fluorescent labels for the detection of aluminium are morin (2′,3,4′,5,7-pentahydroxyflavone) and lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid]. While the former has gained popularity in the detection of aluminium in plants and predominantly within root tips, the latter boasts greater sensitivity and selectivity for the detection of aluminium in human cells and tissues. Herein, we have developed a simplified morin staining protocol using the autofluorescence quenching agent, Sudan Black B. This modified protocol improves tissue morphology and increases analytical sensitivity, which allows intracellular aluminium to be detected in monocytes and when co-localised with senile plaques in human brain tissue of donors diagnosed with familial Alzheimer’s disease. Overall, our results demonstrate a simple approach to minimise false positives in the use of morin to unequivocally detect aluminium in vivo. Electronic supplementary material The online version of this article (10.1007/s00418-019-01809-0) contains supplementary material, which is available to authorized users.

Brain slices have been the workhorse for many neuroscience labs since the pioneering work of Henry McIlwain in the 1950s. Their utility is undisputed and their acceptance as appropriate models for the central nervous system is widespread, if not universal. However, the skeleton in the closet is that ATP levels in brain slices are lower than those found in vivo, which may have important implications for cellular physiology and plasticity. Far from this being a disadvantage, the ATP-impoverished slice can serve as a useful and experimentally-tractable surrogate for the injured brain, which experiences similar depletion of cellular ATP. We have shown that the restoration of cellular ATP in brain slices to in vivo values is possible with a simple combination of d-ribose and adenine (RibAde), two substrates for ATP synthesis. Restoration of ATP in slices to physiological levels has implications for synaptic transmission and plasticity, whilst in the injured brain in vivo RibAde shows encouraging positive results. Given that ribose, adenine, and a third compound, allopurinol, are all separately in use in man, their combined application after acute brain injury, in accelerating ATP synthesis and increasing the reservoir of the neuroprotective metabolite, adenosine, may help reduce the morbidity associated with stroke and traumatic brain injury.\ud \ud

The article deals with testing and evaluating the mould resistance of carbide lime by-product from deposits in comparison with the effect of reference conventional slaked lime. The mould resistance of limes was tested on a mixture of mould such as Gliocladium virens, Paecilomyces variotii, Penicillium funiculosum, Chaetomium globosum, and Aspergillus niger as well as evaluated according to the method stated in the technical standard STN 724310: 1993. The evaluating scale for mould resistance is from 0 up to 5 in the fungal growth degree (FGD). The FGD value 0 stands for no fungal growth comes about and construction materials and products have the microbiostatic effect against filamentous fungi. The experiments confirm the microbiostatic effect against mould of carbide lime by-product from deposits as well as reference conventional slaked lime; however, the microbicidal effect against mould has not been confirmed at all; the inhibiting area-zone-did not form on the agar growth media around the tested samples. Application of carbide lime by-product from deposits ensures increased mould resistance of construction materials with the microbiostatic effect against mould in accordance with technical standard STN 724310: 1993. Web of Science 73 10 2582 2575

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and D-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace-multi-capillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS). The results obtained by SHS-MCC-GC-IMS are compared with those obtained by the more conventional analytical technique of headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.

Intracellular filamentous tau pathology is the defining feature of tauopathies, which form a subset of neurodegenerative diseases. We have analyzed pathological tau in Alzheimer’s disease, and in frontotemporal lobar degeneration associated with tauopathy to include cases with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, and ones due to intronic mutations in MAPT. We found that the C-terminal band pattern of the pathological tau species is distinct for each disease. Immunoblot analysis of trypsin-resistant tau indicated that the different band patterns of the 7–18 kDa fragments in these diseases likely reflect different conformations of tau molecular species. Protein sequence and mass spectrometric analyses revealed the carboxyl-terminal region (residues 243–406) of tau comprises the protease-resistant core units of the tau aggregates, and the sequence lengths and precise regions involved are different among the diseases. These unique assembled tau cores may be used to classify and diagnose disease strains. Based on these results, we propose a new clinicopathological classification of tauopathies based on the biochemical properties of tau. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1503-3) contains supplementary material, which is available to authorized users.

In this study, we sourced 37 commercial flours from 14 mills based on 7 countries and analysed them with a total of 18 methods, generating 90 single analytical values for each flour. The 18 methods were chosen to cover the analytical practice of most European mills and bakery companies, as well as particle charge detection, GlutoPeak and solvent retention capacity as emerging methods. We investigated the relationship between the data from the individual methods, and performed a principal component analysis to describe the structure of the data set and identify the main underlying flour properties. Four principal components accounted for 64.8% of the total variance. They were interpreted as (PC1) starch gelatinization properties, (PC2) hydration properties, (PC3) dough resistance at variable water amount, and (PC4) dough strength at fixed water amount. From the emerging methods, solvent retention capacity (sodium carbonate and water) was highly correlated with PC2, while the GlutoPeak max torque was highly correlated with PC4.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I–II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds., Financial support was provided by theMinisterio de Economía y Competitividad (MINECO), projects FIS/ISCIII PI11/01436 and PI11/00592, MAT2015-65184-C2-2-R, and the CSIC. The research leading to these results was funded by the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 312284 (CALIPSO). Synchrotron measurements were performed at the SMIS beamline of Synchrotron SOLEIL. PS is grateful to MINECO for Ramón y Cajal postdoctoral fellowships (RYC-2014-16759). We would like to thank all the beamline staff for their helpful support and advice.

There is tremendous disparity in scientific productivity among nations, particularly in Latin America. At first sight, this could be linked to the relative economic health of the different countries of the region, but even large and relatively rich Latin American countries do not produce a good level of science. Although Latin America has increased the number of its scientists and research institutions in recent years, the gap between developed countries and Latin American countries is startling. The prime importance of science and technology to the development of a nation remains unacknowledged. The major factors contributing to low scientific productivity are the limited access to grant opportunities, inadequate budgets, substandard levels of laboratory infrastructure and equipment, the high cost and limited supply of reagents, and inadequate salaries and personal insecurity of scientists. The political and economic instability in several Latin America countries results in a lack of long-term goals that are essential to the development of science. In Latin America, science is not an engine of the economy. Most equipment and supplies are imported, and national industries are not given the incentives to produce these goods at home. It is a pity that Latin American society has become accustomed to expect new science and technological developments to come from developed countries rather than from their own scientists. In this article, we present a critical view of the Latin American investigator’s daily life, particularly in the area of biomedicine. Too many bright young minds continue to leave Latin America for developed countries, where they are very successful. However, we still have many enthusiastic young graduates who want to make a career in science and contribute to society. Governments need to improve the status of science for the sake of these young graduates who represent the intellectual and economic future of their countries. Fil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Delgado, Gabriela. Universidad Nacional de Colombia; Colombia

An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar “glyco tune mix”. When subjected to liquid chromatography–ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment. Graphical AbstractDiscrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0235-8) contains supplementary material, which is available to authorized users.

In recent years the benefit of measuring positionally resolved 13C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13C-Metabolic Flux Analysis (13C-MFA) has become evident. However, the usage of positional labeling information for 13C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-0174-9) contains supplementary material, which is available to authorized users.

The purpose of this study was to evaluate the importance of exogenous water intake (snow/seawater) in hooded seal (Cystophora cristata) pups during their post-weaning fast. In this study, five hooded seal pups had ad lib access to snow and seawater for the first 12 and last 21 days of their post-weaning fast, respectively. Total body water and water flux were determined during both exposure periods by use of the tritiated water method. Blood samples were collected to monitor changes in hematocrit, plasma urea and plasma osmolality. Body mass loss was on average 0.36 kg day−1. Average total body water changed from 15.7 to 11.4 L, while total water influx changed from 15 to 18 mL day−1 kg−1 during snow and seawater exposure, respectively. Of this influx an average of 35% can be attributed to metabolic water, while approximately 8% was due to respiratory water influx. Interestingly, 56 and 58% of the total water influx was due to snow and seawater ingestion, respectively, amounting to 8 mL day−1 kg−1 snow (counted as liquid water) and 10 mL day−1 kg−1 seawater. Based on the results of the plasma parameters it is concluded that fasting hooded seal pups maintain water balance and homeostasis when access to snow or seawater is permitted. It is further concluded that snow and seawater intake, in addition to metabolic and respiratory water, is important for maintenance of water balance and excretion of urea during the post-weaning fast of hooded seal pups. Electronic supplementary material The online version of this article (doi:10.1007/s00360-016-1048-3) contains supplementary material, which is available to authorized users.

Time temperature integrators (TTIs) are useful tools in estimating the heat load applied on differently processed dairy products. The objective of this study was to analyze and assess three TTIs – lactulose, furosine, and acid-soluble β-lactoglobulin (β-Lg) – in 70 high heated dairy products at retail in Austria and Germany comprising whipping cream, coffee cream/milk, and condensed milk products. While β-Lg was not appropriate to evaluate the heat load of these products, furosine and especially lactulose increased with rising intensity of heat treatment, and are appropriate to distinguish between several heating categories analyzed. Pasteurized (n = 8) and “heat treated” (n = 5) whipping cream samples showed lowest furosine (48 ± 14/ 45 ± 19 mg.100 g−1 protein) and low lactulose (29 ± 10/57 ± 28 mg.L−1) concentrations, followed by ESL whipping cream (n = 10), ESL coffee cream (n = 1), and UHT whipping cream (n = 10) (furosine = 72 ± 37/71/161 ± 30 mg.100 g−1 protein; lactulose = 56 ± 41/161/195 ± 39 mg.L−1), respectively. Sterilized condensed milk samples (n = 14) showed the highest concentrations of both TTIs and could be clearly separated from UHT treated samples (n = 5) (furosine = 491 ± 196/216 ± 46 mg.100 g−1 protein; lactulose = 1997 ± 658/409 ± 161 mg.L−1), whereas the so-called heat-treated samples (n = 9) had a heat load in between showing an extreme range of variation for both TTIs.

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l(-1), which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria. Electronic supplementary material The online version of this article (doi:10.1007/s11120-007-9264-z) contains supplementary material, which is available to authorized users.

Laboratory-scale anaerobic degradation of monoethanolamine waste (MEAw) with co-substrate organics was conducted at room temperature and organic loading rates from 0.19 to 5.03 kg COD/m3 day for 486 days in a hybrid digester. 90 % feed COD conversion to methane was obtained at the lower loads and only 45 % at the highest MEA waste/COD ratio (MEAwr) of 0.62 due to inhibition of methanogenesis. Inhibition at comparable loads decreased with time, implying that the culture adapted to the challenging feed. Methane yield was negatively correlated to MEAwr applied and inhibition avoided at MEAwr

In the next 10 years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs. Graphical Abstract Experimental approach for analysing resin beads by attenduated total reflection alongside typical FTIR spectra Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8871-3) contains supplementary material, which is available to authorized users.

X-linked adrenoleukodystrophy (X-ALD) is a rare neurometabolic disease characterized by the accumulation of very long chain fatty acids (VLCFAs) due to a loss of function of the peroxisomal transporter ABCD1. Here, using in vivo and in vitro models, we demonstrate that autophagic flux was impaired due to elevated mammalian target of rapamycin (mTOR) signaling, which contributed to X-ALD pathogenesis. We also show that excess VLCFAs downregulated autophagy in human fibroblasts. Furthermore, mTOR inhibition by a rapamycin derivative (temsirolimus) restored autophagic flux and inhibited the axonal degenerative process as well as the associated locomotor impairment in the Abcd1−/Abcd2−/− mouse model. This process was mediated through the restoration of proteasome function and redox as well as metabolic homeostasis. These findings provide the first evidence that links impaired autophagy to X-ALD, which may yield a therapy based on autophagy activators for adrenomyeloneuropathy patients. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1378-8) contains supplementary material, which is available to authorized users.

Increasing temperatures and glacier melting at the Western Antarctic Peninsula (WAP) are causing rapid changes in shallow coastal and shelf systems. Climate change-related rising water temperatures, enhanced ice scouring, as well as coastal sediment runoff, in combination with changing feeding conditions and microbial community composition, will affect all elements of the nearshore benthic ecosystem, a major component of which is the Antarctic soft-shelled clam Laternula elliptica. A 454-based RNA sequencing was carried out on tissues and hemocytes of L. elliptica, resulting in 42,525 contigs, of which 48 % was assigned putative functions. Changes in the expression of putative stress response genes were then investigated in hemocytes and siphon tissue of young and old animals subjected to starvation and injury experiments in order to investigate their response to sedimentation (food dilution and starvation) and iceberg scouring (injury). Analysis of antioxidant defense (Le-SOD and Le-catalase), wound repair (Le-TIMP and Le-chitinase), and stress and immune response (Le-HSP70, Le-actin, and Le-theromacin) genes revealed that most transcripts were more clearly affected by injury rather than starvation. The upregulation of these genes was particularly high in the hemocytes of young, fed individuals after acute injury. Only minor changes in expression were detected in young animals under the selected starvation conditions and in older individuals. The stress response of L. elliptica thus depends on the nature of the environmental cue and on age. This has consequences for future population predictions as the environmental changes at the WAP will differentially impact L. elliptica age classes and is bound to alter population structure.

Our previous studies carried out on the pilocarpine model of seizures showed that highly resolved elemental analysis might be very helpful in the investigation of processes involved in the pathogenesis of epilepsy, such as excitotoxicity or mossy fiber sprouting. In this study, the changes in elemental composition that occurred in the hippocampal formation in the electrical kindling model of seizures were examined to determine the mechanisms responsible for the phenomenon of kindling and spontaneous seizure activity that may occur in this animal model. X-ray fluorescence microscopy was applied for topographic and quantitative analysis of selected elements in tissues taken from rats subjected to repetitive transauricular electroshocks (ES) and controls (N). The detailed comparisons were carried out for sectors 1 and 3 of the Ammon’s horn (CA1 and CA3, respectively), the dentate gyrus (DG) and hilus of DG. The obtained results showed only one statistically significant difference between ES and N groups, namely a higher level of Fe was noticed in CA3 region in the kindled animals. However, further analysis of correlations between the elemental levels and quantitative parameters describing electroshock-induced tonic and clonic seizures showed that the areal densities of some elements (Ca, Cu, Zn) strongly depended on the progress of kindling process. The areal density of Cu in CA1 decreased with the cumulative (totaled over 21 stimulation days) intensity and duration of electroshock-induced tonic seizures while Zn level in the hilus of DG was positively correlated with the duration and intensity of both tonic and clonic seizures.

Intrinsically disordered proteins (IDPs) refer to those proteins without fixed three-dimensional structures under physiological conditions. Although experiments suggest that the conformations of IDPs can vary from random coils, semi-compact globules, to compact globules with different contents of secondary structures, computational efforts to separate IDPs into different states are not yet successful. Recently, we developed a neural-network-based disorder prediction technique SPINE-D that was ranked as one of the top performing techniques for disorder prediction in the biannual meeting of critical assessment of structure prediction techniques (CASP 9, 2010). Here, we further analyze the results from SPINE-D prediction by defining a semi-disordered state that has about 50 % predicted probability to be disordered or ordered. This semi-disordered state is partially collapsed with intermediate levels of predicted solvent accessibility and secondary structure content. The relative difference in compositions between semi-disordered and fully disordered regions is highly correlated with amyloid aggregation propensity (a correlation coefficient of 0.86 if excluding four charged residues and proline, 0.73 if not). In addition, we observed that some semi-disordered regions participate in induced folding, and others play key roles in protein aggregation. More specifically, a semi-disordered region is amyloidogenic in fully unstructured proteins (such as alpha-synuclein and Sup35) but prone to local unfolding that exposes the hydrophobic core to aggregation in structured globular proteins (such as SOD1 and lysozyme). A transition from full disorder to semi-disorder at about 30–40 Qs is observed in the poly-Q (poly-glutamine) tract of huntingtin. The accuracy of using semi-disorder to predict binding-induced folding and aggregation is compared with several methods trained for the purpose. These results indicate the usefulness of three-state classification (order, semi-disorder, and full-disorder) in distinguishing nonfolding from induced-folding and aggregation-resistant from aggregation-prone IDPs and in locating weakly stable, locally unfolding, and potentially aggregation regions in structured proteins. A comparison with five representative disorder-prediction methods showed that SPINE-D is the only method with a clear separation of semi-disorder from ordered and fully disordered states.

Neoadjuvant chemotherapy is used in patients with locally advanced breast cancer to reduce the tumor size before surgery. Unfortunately, resistance to chemotherapy may arise from a variety of mechanisms. The heat shock proteins (HSPs), which are highly expressed in mammary tumor cells, have been implicated in anticancer drug resistance. In spite of the widely described value of HSPs as molecular markers in cancer, their implications in breast tumors treated with anthracycline-based neoadjuvant chemotherapy has been poorly explored. In this study, we have evaluated by immunohistochemistry the expression of HSP27 (HSPB1) and HSP70 (HSPA) in serial biopsies from locally advanced breast cancer patients (n= 60) treated with doxorubicin (DOX) or epirubicin (EPI) based monochemotherapy. The serial biopsies were taken at days 1, 3, 7, 21, and compared with the pre-chemotherapy and the surgical biopsies. After surgery the patients received additional chemotherapy with Cyclophosphamide, Methotrexate and 5-Fluorouracil (CMF). High nuclear HSPB1 and HSPA expressions were found in invasive cells after DOX/EPI administration (P31% of the cells) and cytoplasmic HSPA expressions (>11% of the tumor cells) were associated with better DFS (P=0.0348 and P=0.0118, respectively). We conclude that HSPA expression may be a useful prognostic marker in breast cancer patients treated with neoadjuvant DOX/EPI chemotherapy, indicating the need to change the administered drugs after surgery for overcoming drug resistance. Fil: Nadin, Silvina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Sottile Fleury, Mayra Lis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Montt Guevara, Maria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Gauna, Gisel Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Daguerre, Pedro. Hospital Luis Lagomaggiore; Argentina Fil: Leuzzi, Marcela. Hospital Luis Lagomaggiore; Argentina Fil: Gago, Francisco E.. Universidad Nacional de Cuyo; Argentina Fil: Ibarra, Jorge. Hospital Luis Lagomaggiore; Argentina Fil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Vargas Roig, Laura Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina