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Graphical abstract During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03317-4.

Background Lysine succinylation is a naturally occurring post-translational modification (PTM) that is ubiquitous in organisms. Lysine succinylation plays important roles in regulating protein structure and function as well as cellular metabolism. Global lysine succinylation at the proteomic level has been identified in a variety of species; however, limited information on lysine succinylation in plant species, especially paper mulberry, is available. Paper mulberry is not only an important plant in traditional Chinese medicine, but it is also a tree species with significant economic value. Paper mulberry is found in the temperate and tropical zones of China. The present study analyzed the effects of lysine succinylation on the growth, development, and physiology of paper mulberry. Results A total of 2097 lysine succinylation sites were identified in 935 proteins associated with the citric acid cycle (TCA cycle), glyoxylic acid and dicarboxylic acid metabolism, ribosomes and oxidative phosphorylation; these pathways play a role in carbon fixation in photosynthetic organisms and may be regulated by lysine succinylation. The modified proteins were distributed in multiple subcellular compartments and were involved in a wide variety of biological processes, such as photosynthesis and the Calvin-Benson cycle. Conclusion Lysine-succinylated proteins may play key regulatory roles in metabolism, primarily in photosynthesis and oxidative phosphorylation, as well as in many other cellular processes. In addition to the large number of succinylated proteins associated with photosynthesis and oxidative phosphorylation, some proteins associated with the TCA cycle are succinylated. Our study can serve as a reference for further proteomics studies of the downstream effects of succinylation on the physiology and biochemistry of paper mulberry.

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

This paper proposes a combined strategy of using paper-based competitive immunochromatography and a near field communication (NFC) tag for wireless cotinine determination. The glucose oxidase labeled cotinine antibody specifically binds free cotinine in a sample, whereas the unoccupied antibody attached to BSA-cotinine at the test line on a lateral flow strip. The glucose oxidase on the strip and an assistant pad in the presence of glucose generated H2O2 and imposed the Ag oxidation on the modified electrode. This enabled monitoring of immunoreaction by either electrochemical measurement or wireless detection. Wireless sensing was realized for cotinine in the range of 100–1000 ng/mL (R2 = 0.96) in PBS medium. Undiluted urine samples from non-smokers exhibited an Ag-oxidation rate three times higher than the smoker’s urine samples. For 1:8 diluted urine samples (smokers), the proposed paper-based competitive immunochromatography coupled with an enzyme-modified electrode differentiated positive and negative samples and exhibited cotinine discrimination at levels higher than 12 ng/mL. This novel sensing platform can potentially be combined with a smartphone as a reader unit.

A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 &times, 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.

Made available in DSpace on 2022-04-29T08:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 This work addresses a method that combines immunomagnetic separation (IMS) and paper-based nucleic acid immunochromatographic assay for the sensitive detection of Mycolicibacterium fortuitum (basonym Mycobacterium fortuitum) In particular, the preconcentration of the bacteria was achieved by using magnetic particles modified with an antibody specific towards mycobacteria. Following the IMS, the bacteria were lysed, and the genome was amplified by double-tagging PCR, using a set of primers specific for the 16S rRNA gene for Mycobacterium. During the amplification, the amplicons were labeled with biotin and digoxigenin tags. Moreover, a comparative study of paper-based immunochromatographic platforms, relying on vertical and lateral flow and on the use of streptavidin gold nanoparticles as a signal generating system, was also performed. The visual readout was achieved when the gold-modified amplicons were captured by the anti-DIG antibody in the test line. The analytical performance of both methods, nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF), is also discussed. Although NALF showed lower limit of detections (LODs), both NALF and NAVF combined with IMS were able to detect the required LOD in hemodialysis water, becoming two promising and useful techniques for the rapid screening of water supplies in hemodialysis centers, to prevent the exposure of immunosuppressed patients to contaminated sources. Departament de Química Universitat Autònoma de Barcelona Department of Analytical Chemistry Institute of Chemistry São Paulo State University Departament de Genètica i de Microbiologia Facultat de Biociències Universitat Autònoma de Barcelona Institute of Biotechnology and Biomedicine Universitat Autònoma de Barcelona Department of Analytical Chemistry Institute of Chemistry São Paulo State University

The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as Human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 10(2) copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.

As a lignocellulosic material, wastepaper is a potential material for ethanol production. However, little research on the enzymatic hydrolysis of wastepaper pulp has been conducted. In this study, the enzymatic hydrolysis of different waste pulp fractions (R80 represents greater than 80-mesh wastepaper pulp, R80-180 represents the range of 80- to 180-mesh wastepaper pulp, and R180 represents smaller than 180-mesh waste paper pulp) were carried out at 50 °C, pH 4.8, for 96 h, with a substrate concentration of 5% (w/v) and cellulase loading of 18 FPU/g cellulose. In terms of the specific surface area, fiber structure, and surface morphology, R80-180 had the highest affinity to cellulase and therefore the highest glucose yield of 80.33%. R180 had the lowest glucose yield (55.36%) because of its high ash content (21.36%), which reduced the adsorption of cellulase to cellulose. The enzymatic hydrolysis of R80 mixed with R80 or R80-180 was also studied. Results indicated that adding R80-180 increased the glucose yield of R80. The highest glucose yield (82.57%) was obtained when 15% R80-180 was mixed with R80. However, the glucose content decreased when R180 was mixed with R80 because of its high ash content.

International audience; The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.

The antioxidant effects of the ethyl acetate fraction of Dillenia indica bark (DIBEt) and the underlying mechanisms were investigated in tert-butyl hydroperoxide (t-BHP)-stimulated oxidative stress in RAW 264.7 cells. Paper spray ionization-mass spectroscopy with positive-ion mode tentatively revealed 27 secondary metabolites in D. indica bark extract, predominant among them were alkaloids, phenolic acids, and flavonoids. A new triterpenoid (nutriacholic acid) was confirmed in DIBEt for the first time. DIBEt had strong free radical-scavenging capabilities and was also able to reduce t-BHP-induced cellular reactive oxygen species (ROS) generation in RAW 264.7 cells. DIBEt was found to prevent oxidative stress by boosting the levels of heme oxygenase-1 (HO-1) through the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) via the regulation of extracellular signal-regulated kinase (ERK) phosphorylation in RAW 264.7 cells. These results support the potential of DIBEt for defense against oxidative stress-stimulated diseases.

Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority (www.prionpriority.eu) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper (www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies. © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC.

Tuberculosis (TB) remains one of the most serious infectious diseases in the world and the rate of new cases continues to increase. The development of cheap and simple methodologies capable of identifying TB causing agents belonging to the Mycobacterium tuberculosis Complex (MTBC), at point-of-need, in particular in resource-poor countries where the main TB epidemics are observed, is of paramount relevance for the timely and effective diagnosis and management of patients. TB molecular diagnostics, aimed at reducing the time of laboratory diagnostics from weeks to days, still require specialised technical personnel and labour intensive methods. Recent nanotechnology-based systems have been proposed to circumvent these limitations. Here, we report on a paper-based platform capable of integrating a previously developed Au-nanoprobe based MTBC detection assay—we call it “Gold on Paper”. The Au-nanoprobe assay is processed and developed on a wax-printed microplate paper platform, allowing unequivocal identification of MTBC members and can be performed without specialised laboratory equipment. Upon integration of this Au-nanoprobe colorimetric assay onto the 384-microplate, differential colour scrutiny may be captured and analysed with a generic “smartphone” device. This strategy uses the mobile device to digitalise the intensity of the colour associated with each colorimetric assay, perform a Red Green Blue (RGB) analysis and transfer relevant information to an off-site lab, thus allowing for efficient diagnostics. Integration of the GPS location metadata of every test image may add a new dimension of information, allowing for real-time epidemiologic data on MTBC identification.

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely ‘TB Detect’ (containing BioFM-Filter device), ‘TB Concentration and Transport’ (containing Trans-Filter device) and ‘TB DNA Extraction’ kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (‘‘cells-in-gels-in-paper’’ or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, ‘‘sections’’ all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

Mass production of vermicompost using suitable species of earthworms and selecting target organic waste materials has appeared to be a great development in the realm of biotechnological research for the sustainable eco-management. Although, for the bioconversion of organic wastes to vermicompost, suitable earthworm species play major roles, a hoard of bacterial assemblages by virtue of production of different enzymes facilitate the process of vermicomposting. The present study has documented the roles of vermicompost associated bacteria in combating, preventing, and controlling of cancer so as to open a new vista not only in the field of vermitechnology but also on biomedical research. Earthworms’ associated bacterial metabolic products having their unique physicochemical excellence have gained importance due to their roles as a facilitator of apoptosis (programed cell death in a MCF-7 cell line). The antioxidant and anticancer activities of ethyl acetate extracts’ of vermicompost associated bacteriumBacillus anthraciswere undertaken by antioxidant assay which revealed maximum DPPH radical scavenging effect (75.79 ± 5.41%) of the extracts’ at 9 00 μg ml-1. Furthermore, the crude extracts obtained from the same bacteria were found to decrease the activity of SOD (superoxide dismutase) with the increase in doses. MTT assay showed potent cytotoxic activity against human breast adenocarcinoma cells (MCF-7) with the IC50 value of 46.64 ± 0.79 μg ml-1. It was further confirmed through Hoechst 33258 staining of nuclear fragmentation assay and DNA fragmentation analysis. Western blotting test has confirmed a downregulation of Akt upon application of crude extracts. Increase of SOD activity along with decrease of Akt level reflects that the mode of action is entirely PI-3K dependent. This study tends to indicate thatB.anthracisisolated from vermicompost could be potentially explored for the development of new therapeutic agents, especially against cancer.

aDepartment of Molecular Microbiology, CIB, Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, E-28006 Madrid, Spain; bDepartment of Textile and Paper Engineering, ETSII, Polytechnic University of Catalunya (UPC), Colón 11, E-08222 Terrassa, Spain; cDepartment of Biogeochemistry, IRNAS, CSIC, PO Box 1052, E-41080, Seville, Spain; dCelulosas de Levante SA (CELESA), Ctra. C-237, km 4.5, E-43500 Tortosa, Spain, High-quality flax pulp was bleached in a totally-chlorine-free (TCF) sequence using a laccase-mediator system. Three fungal laccases (from Pycnoporus cinnabarinus, Trametes versicolor and Pleurotus eryngii) and two mediators, 2,2´-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 1-hydroxybenzotriazole (HBT), were compared. P. cinnabarinus and T. versicolor laccases in the presence of HBT gave the best results in terms of high brightness and low lignin content (kappa number). The former laccase also resulted in the best preservation of cellulose and the largest removal of residual lignin as revealed by analytical pyrolysis, and was selected for subsequent TCF bleaching. Up to 90% delignification and strong brightness increase were attained after a laccase-mediator treatment followed by H2O2 bleaching. This TCF sequence was further improved by applying H2O2 under pressurized O2. In this way, we obtained up to 82% ISO brightness (compared with 37% in the initial pulp, and 60% in the peroxide-bleached control) and very low kappa number (near 1). Good results were also found when the laccase-mediator treatment was performed in a bioreactor under pressurized oxygen. The pulp properties obtained, which could not be attained by conventional TCF bleaching of flax pulp, demonstrate the feasibility of enzymatic bleaching to substitute chlorine-containing reagents in manufacturing of these high-price paper pulps., This study was supported by the European Commission (contract QLK3-99-590), the Spanish CICYT (FEDER project 2FD97-0896-C02, and projects AGL2002-00393, BIO2002-1166 and PPQ2000-1068-C02-02), the Comunidad de Madrid, and the CSIC network on “Bioremediation and Phytoremediation”.

aCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain bInstituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1042, E-41080 Sevilla, Spain cENCE, CIT, Carretera de Campañó s/n, E-36157 Pontevedra, Spain, In the context of environmentally-friendly bleaching in paper pulp manufacturing, an enzymatic procedure was developed for the isolation of residual lignins from eucalypt pulp. The method was based on the hydrolysis and complete solubilization of pulp cellulose using a combination of Trichoderma cellulase and Aspergillus beta-glucosidase, followed by lignin purification using Bacillus protease (for hydrolysis of contaminating cellulase) and extraction of lignin with dimethylacetamide and NaOH. This method was applied to both unbleached and totally chlorine free bleached eucalypt kraft pulps. The different fractions obtained were analyzed by Fourier-transform infrared spectroscopy and pyrolysis-gas chromatography/mass spectrometry to evaluate the effect of the different isolation steps, and optimize the lignin isolation procedure. Depending of the type of pulp and its delignification degree most of the lignin was recovered from the cellulase hydrolyzate or the non-hydrolyzable residue. High-purity lignins were obtained from pulps by a new isolation procedure that included double solvent purification after combining the two fractions from protease treatment. This method is being used to analyze the effect of chlorine-free reagents (including enzymes) in clean bleaching sequences., This study has been supported by ENCE, the EU-Contract QLK5-99-1357, and the Spanish project AGL2002-00393.

A rhodium(III) complex, [Rh(MQ)(DMSO)2Cl2] (1), with 8-hydroxy-2-methylquinoline as the ligand was synthesized and characterized. Complex 1 exhibited cytotoxicity against BEL-7404, Hep-G2, NCI-H460, T-24, and A549 cell lines with IC50 values in the micromolar range (6.52–17.86 μM). Various experiments on the Hep-G2 cells showed that complex 1 caused cell cycle arrest at the S phase, downregulation of cdc25 A, cyclin A, cyclin B and CDK2, and upregulation of p21, p27 and p53. Furthermore, cytotoxicity mechanism studies suggested that complex 1-induced apoptosis was achieved via disruption of the mitochondrial function, which led to a significant loss of the mitochondrial membrane potential, an increase in the cellular levels of reactive oxygen species, cytochrome c, and apaf-1, and a fluctuation of the intracellular Ca2+ concentration. Taken altogether, complex 1 can trigger cancer cell death by inducing apoptosis through a mitochondrial dysfunction pathway.

Chlorinated dehydroabietic acids are formed during the chlorine bleaching of wood pulp and are very toxic to fish. Thus, destruction of these compounds is an important function of biological treatment systems for pulp and paper mill effluents. In this study, 12 strains of diverse, aerobic resin acid-degrading bacteria were screened for the ability to grow on chlorinated dehydroabietic acids as sole organic substrates. All seven strains of the class Proteobacteria able to use dehydroabietic acid were also able to use a mixture of 12- and 14-chlorodehydroabietic acid (Cl-DhA). None of the strains used 12,14-dichlorodehydroabietic acid. Sphingomonas sp. strain DhA-33 grew best on Cl-DhA and simultaneously removed both Cl-DhA isomers. Ralstonia sp. strain BKME-6 was typical of most of the strains tested, growing more slowly on Cl-DhA and leaving higher residual concentrations of Cl-DhA than DhA-33 did. Strains DhA-33 and BKME-6 mineralized (converted to CO(inf2) plus biomass) 32 and 43%, respectively, of carbon in Cl-DhA consumed. Strain DhA-33 produced a metabolite from Cl-DhA, tentatively identified as 3-oxo-14-chlorodehydroabietin, and both strains produced dissolved organic carbon which may include unidentified metabolites. Cl-DhA removal was inducible in both DhA-33 and BKME-6, and induced DhA-33 cells also removed 12,14-dichlorodehydroabietic acid. Based on activities of strains DhA-33 and BKME-6, chlorinated DhAs, and potentially toxic metabolite(s) of these compounds, are relatively persistent in biological treatment systems and in the environment.

Due to rapid depletion of wild stocks, the necessity to cultivate fish is eminent. Current fish farming practices seek to improve flesh quality. The notion that white muscles are the main target of the fishing industry is emphasized. A novel approach is suggested based on the development of white muscles in wild fish from eggs to adults. A compilation of facts about white muscle structure, function and ontogeny is followed by an account of the changes in swimming behaviour and performance related to the use of white muscle during growth from larva to adult. Ecological data narrate early swimming performance with white muscle development and growth, unveiling some of the important natural selection factors eliminating weak swimmers and poor growers from the breeding stock. A comparison between fish culture practise and natural conditions reveals fundamental differences. New approaches following wild breeding processes promise several important advantages regarding the quality of white muscle.

16 páginas, 15 figuras, 2 tablas.-- et al., We report that hydrophilic oxidized multiwalled carbon nanotube papers (CNPs) were efficient substrates for attachment, spreading, and proliferation of MG-63 cells. Complete spreading occurred in the first 4 h of culture even without complete serum or pre-adsorbed adhesion proteins such as collagen or fibronectin. By contrast, the density of adherent cell on hydrophobic CNPs was low. Spreading did not happen after 24 h of culture in absence of serum proteins and growth factors on such papers. Cell viability concomitantly decreased. We also observed that a short RGD peptide, designed to adsorb on both hydrophobic and hydrophilic CNPs, enhanced adhesion, spreading, and proliferation only on hydrophilic CNPs. By contrast, adsorbed fibronectin triggers cell adhesion and spreading on both types of CNPs. Our results show that the imitation of tissue basal membranes with the nanofibrous structure of CNPs is insufficient to trigger the proliferation and spread of cells. Chemical cues are also needed for cells to spread. CNPs, however, offer a better substrate for adhesion protein absorption than do non-porous polymers.

Early last summer Protein Science received a manuscript by Michael J. Behe and David W. Snoke entitled, “Simulating Evolution by Gene Duplication of Protein Features That Require Multiple Amino Acid Residues.” The editors were aware that the authors of the paper are associated with the “Intelligent Design” movement, which challenges Darwinian evolutionary theory on the grounds that not enough time has elapsed since life arose on Earth to account for the complexity of living organisms and the molecules they contain without the intervention of an unnamed “designer.” While these associations with an idea that clearly steps over the bounds of what can be determined by scientific means put us in an uncomfortable position, the editors, in the interest of fairness, sent the manuscript to reviewers in the usual way. Two extensive reviews were obtained, which asked for changes to the manuscript. The authors addressed the concerns in the ways prescribed by the journal, and the paper was published in the October 2004 issue (Behe and Snoke 2004). Needless to say, it drew a great deal of attention from our usual audience and other scientists in evolutionary studies. The letters we received contained many points of disagreement with the Behe and Snoke article, some of which are summarized below. In addition, Professor Michael Lynch of Indiana University indicated to me that the article raised some issues that Dr. Lynch was considering and asked if we would be interested in a response to the Behe and Snoke article in the form of a research manuscript. At that point in the proceedings, it was not clear to me what the best approach would be, so I indicated to all parties that we would consider letters and responses, and I also indicated that we would welcome the study by Prof. Lynch as a submission to the journal. I indicated that Behe and Snoke would be invited to respond to anything we were considering for publication. All parties were informed that everything to be considered for publication in the journal, whether in letter or manuscript form, would have to go through careful scientific review and receive endorsement in order to be published. Prof. Lynch’s paper, “Simple Evolutionary Pathways to Complex Proteins,” was received late last year and sent to reviewers. They returned reviews requesting a few changes that were subsequently addressed by Dr. Lynch. Drs. Behe and Snoke then sent a lengthy response to Dr. Lynch’s article entitled, “A Response to Michael Lynch.” The essence of the response was that Behe and Snoke assume that intermediate mutations on the way to a new multi-residue feature are inevitably deleterious, while Lynch allows for neutrality or positive selection. Thus, the response reiterated the model and positions of the October 2004 paper. The various letters and manuscripts were reviewed as a package by the editors and by outside experts. Some points made by the various commentators include: Substantial variation in the rate of mutation fixation occurs, both between lineages and between sites on a protein during evolution. This is a central concept of modern population genetics (Fitch and Markowitz 1970; Fitch 1971; Kimura 1983; Yang 1996; Whelan and Goldman 2004). Changes in one site are known to cause changes in the mutation and acceptance rate at other sites in a protein, generally called “compensatory” changes (Fitch and Markowitz 1970; Fitch 1971; Altschuh et al. 1987; Pollock et al. 1999). Recombination strongly accelerates the rate of joining of independent mutations at multiple sites and of grafting new domains with additional functions and sites of interaction to proteins to create new modes of action or regulation (de Souza et al. 1996; Teichmann et al. 1998; Thornton and DeSalle 2000; Vogel et al. 2005). Selection acts continuously, and cumulative effects, rather than a single strongly adaptive change, are the basis of evolution under a Darwinian model. Thus, intermediate states must also be assumed to be selected. It was clear when all of the correspondence was analyzed by the editors and reviewers that no useful purpose would be served by opening this topic up to “Letters,” since there was no real dialog, just statements of position. Prof. Lynch’s approach of testing the problem raised by Drs. Behe and Snoke within the modern framework of evolutionary biology represents the desirable scientific approach (Lynch 2005, this issue; Behe and Snoke 2005, this issue). As Bruce Alberts wrote in a Letter to the Editor of the New York Times (Feb. 12, 2005): “In evolution, as in all areas of science, our knowledge is incomplete. But the entire success of the scientific enterprise has depended on an insistence that these gaps be filled by natural explanations, logically derived from confirmable evidence.”

We revisit an old Russian paper by V.V. Dynnik, R. Heinrich and E.E. Sel'kov (1980a,b) describing: "A mathematical model of carbohydrate energy metabolism. Interaction between glycolysis, the Krebs cycle and the H-transporting shuttles at varying ATPases load". We analyse the model mathematically and calculate the control coefficients as a function of ATPase loads. We also evaluate the structure of the metabolic network in terms of elementary flux modes. We show how this model can respond to an ATPase load as well as to the glucose supply. We also show how this simple model can help in understanding the articulation between the major blocks of energetic metabolism, i.e. glycolysis, the Krebs cycle and the H-transporting shuttles.

p21WAF1/Cip1 belongs to the Cip/Kip family of cyclin kinase inhibitors (CKI) (p21Waf1/Cip1, p27Kip1, p57Kip1). p21Waf1/Cip1 was first described as a potent and universal inhibitor of cyclin-dependent kinases (Cdks). Two forms of functionally active p21 has been localized and described: a nuclear and a cytoplasmic forms. In this paper the structures of p21 has been described and as well as it functional biology in terms of differentiation, proliferation and apoptotic activities. The upregulation of p21 by p53-dependent and p53-independent mechanisms is also described., peer-reviewed

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10 10 to 2.6 × 10 12 (average, 7 × 10 11 ) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10 12 cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10 10 to 1.5 × 10 12 (average, 4 × 10 11 ) cells per g (dry weight) of slime was obtained.

The paper Determination of Aldehyde Dehydrogenase (ALDH) Isozymes in Human Cancer Samples – Comparison of Kinetic and Immunochemical Assays, by Piotr Wroczynski*, Jacek Wierzchowski, Jakub Golab, Magdalena Blazejczyk and Dorota Borecka, originally scheduled for publication in this special issue, was already published in an earlier one: Molecules 2002, 7, 896-901.

Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M – H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans. Graphical abstract

A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full‐scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pK i values) in the low micromolar to sub‐micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence‐based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner., Reliable and effective: GlyT1 and GlyT2 MS Binding Assays were used for a library screening investigating 2439 compounds. For GlyT1 and GlyT2 several inhibitors were identified, of which GlyT2 Inhibitors exhibiting excellent fluorescent properties were of great interest. The outcome of this study demonstrated that MS Binding Assays are powerful tools for the examination of compound libraries of reasonable size in a straightforward manner.

This paper describes a method for determining an atomic model of a protein complex using moderate-resolution cryoEM data and distance predictions from deep learning., Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4–8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 Å resolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.

2‐O‐Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2‐O‐glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site‐directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21‐fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2‐position of the substrate, while retaining its activity for several days at elevated temperatures., Osmolyte synthesis at elevated temperatures: 2‐O‐Glucosylglycerol is produced on industrial scale using the promiscuous transglycosylation activity of sucrose phosphorylase. We engineered the catalytic efficiency and regioselectivity of a sucrose phosphorylase that is far more thermostable than the one currently used in industry.

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes., Linalool is a monoterpenoid used as fragrance ingredient, and a promising source for alternative fuels. Here we explore the artificial isoprenol utilization pathway to produce linalool in Escherichia coli using the linalool/nerolidol synthase from Streptomyces clavuligerus. The generation of improved bLinS variants together with substrate and strain optimization increased linalool production and decreased nerolidol by‐product levels.

SARS‐CoV‐2 is the coronavirus responsible for the COVID‐19 pandemic. Proteases are central to the infection process of SARS‐CoV‐2. Cleavage of the spike protein on the virus's capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS‐CoV‐2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS‐CoV‐2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID‐19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated., PDB Code(s): 6VYB, 4Z2A, 5F02, 4P6E, 5TUN, 2IPP and 3H6S

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc‐binding group (ZBG) variants of this thiol‐derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development., Stable “pathoblocker”: A series of compounds with non‐thiol (stable) zinc‐binding groups has been synthesised and tested for inhibition of the collagenase ColH, a key mediator of clostridial pathogenicity. The most promising compound, a phosphonate, was studied for selectivity over potential human off‐targets and its toxicity both in vitro and in vivo, and was shown to significantly reduce collagenase activity.

The mycobacterial PQS dioxygenase AqdC, a cofactor‐less protein with an α/β‐hydrolase fold, inactivates the virulence‐associated quorum‐sensing signal molecule 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti‐virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2 °C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC‐G40K‐A134L‐G220D‐Y238W and AqdC‐G40K‐G220D‐Y238W showed extended half‐lives and the best trade‐off between stability and activity, with increases in Tappm of 11.8 and 6.1 °C and relative activities of 22 and 72 %, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site., Swings and roundabouts: The dioxygenase AqdC, an α/β‐hydrolase fold protein, inactivates a virulence‐associated signal molecule of the bacterial pathogen Pseudomonas aeruginosa. Using computational approaches to predict and engineer thermostable AqdC variants, we found that gains in thermostability (up to +11.8 °C) were traded against losses in activity, likely due to difficulties in opening the entrance to the active site.

Summary The (maximum) growth rate (µmax) hypothesis predicts that cellular and tissue phosphorus (P) concentrations should increase with increasing growth rate, and RNA should also increase as most of the P is required to make ribosomes.Using published data, we show that though there is a strong positive relationship between the µmax of all photosynthetic organisms and their P content (% dry weight), leading to a relatively constant P productivity, the relationship with RNA content is more complex.In eukaryotes there is a strong positive relationship between µmax and RNA content expressed as % dry weight, and RNA constitutes a relatively constant 25% of total P. In prokaryotes the rRNA operon copy number is the important determinant of the amount of RNA present in the cell. The amount of phospholipid expressed as % dry weight increases with increasing µmax in microalgae. The relative proportions of each of the five major P‐containing constituents is remarkably constant, except that the proportion of RNA is greater and phospholipids smaller in prokaryotic than eukaryotic photosynthetic organisms. The effect of temperature differences between studies was minor.The evidence for and against P‐containing constituents other than RNA being involved with ribosome synthesis and functioning is discussed.