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At least 775 records · Page 43

An in-depth understanding of biomass recalcitrance using natural poplar variants as the feedstock

Here, in an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13 C solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons’ stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2 mg g$–1\atop{biomass}$ as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31 P NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor.

09 BIOMASS FUELS↗

Negative Emission Energy Production Technologies: A Techno‐Economic and Life Cycle Analyses Review

Negative emission technologies (NETs) are of growing importance for society to meet the atmospheric carbon levels required to maintain global temperatures under sustainable limits. A wide range of NETs have been proposed, but there are limited NET assessments that integrate life cycle analysis (LCA) and techno‐economic analysis (TEA). This Review gathers NET TEA/LCA findings and compares their costs and greenhouse gas emissions. Eight different NET‐producing transportation fuels and power‐generation technologies are considered: anaerobic digestion (lignocellulosic), fermentation, torrefaction, combustion, fast pyrolysis, gasification, and hydrothermal liquefaction. Most of these technologies sequester carbon either as carbon dioxide or as biochar. Some are carbon negative by virtue of avoided or displaced emissions. Overall, results indicate that NET energy production costs range between $20 and $80 GJ −1 and have emissions from −400 to 100 kg CO 2,eq GJ −1 . These results suggest that there are potential tradeoffs to consider when developing NETs. These results also show that future TEA/LCA studies of NETs are needed to decrease their uncertainty and improve their technology readiness level.

Li, Wenqin↗

Total Recoverable Aluminum: Not Totally Relevant for Water Quality Standards

Abstract A large water quality data set, representing more than 100 surface-water locations sampled from 2007 to 2017 in the Los Alamos area of New Mexico, USA's Pajarito Plateau, was assembled to evaluate Al concentrations in unfiltered and filtered samples. Aluminum concentrations often exceeded United States Environmental Protection Agency (USEPA) and New Mexico ambient water quality criteria (AWQC), regardless of filter size and sample location. However, AWQC are based on laboratory toxicity studies using soluble Al salts and do not reflect natural conditions in Pajarito Plateau surface waters. The plateau is predominately covered by glassy and recrystallized volcanic ashes (e.g., Bandelier Tuff) containing colloidal to sand-sized aluminosilicates. Samples from natural background drainages and areas downstream of developed regions exhibited similar Al concentrations, suggesting that AWQC exceedances are caused by naturally elevated Al concentrations. Solubility calculations indicated that most samples were oversaturated with respect to amorphous Al(OH)3(s). Therefore, AWQC exceedances are likely artifacts of the “total recoverable” sample preparation, which includes acidification and partial digestion, thereby liberating nonbioavailable Al from aluminosilicates. Accordingly, Al concentrations were strongly associated with suspended sediment concentrations (SSCs), implying that aluminosilicates in suspended sediment contributed to AWQC exceedances and Al oversaturation. Solid-phase particle characterization, using X-ray diffraction (XRD) and scanning electron microscopy with electron dispersive spectroscopy (SEM/EDS) did not identify potentially bioavailable amorphous Al(OH)3(s) in any sample tested. Thus, current sample collection and analysis protocols should not be used to evaluate attainment of Al AWQC on the Pajarito Plateau or locations where aluminosilicates are substantial contributors to total recoverable Al. A sample preparation method (e.g., pH 4 extraction) capable of differentiating nonbioavailable and bioavailable forms of Al is recommended. Otherwise, current New Mexico and USEPA sample preparation approaches will continue to generate artifactual AWQC exceedances in surface waters that contain aluminosilicates. Integr Environ Assess Manag 2019;00:1–14. © 2019 SETAC

Ryan, Adam C.↗

Improving the bioaccessibility of lipophilic ingredient in its oral intestinal delivery by ultrasound and biological cross‐linker

Abstract BACKGROUND Great efforts have been made to improve the oral bioaccessibility of lipophilic ingredients with multi‐functionalities. Achieving intestinal delivery of lipophilic ingredients and their encapsulation in micelles composed of bile salts and lipid hydrolysates (i.e. fatty acids) is critical for improving oral bioaccessibility. Therefore, oil‐core microcapsules are considered ideal carriers of lipophilic ingredients. Previous studies have reported oil‐core/zein‐shell microcapsules constructed by a one‐step anti‐solvent process. Still, its efficacy as an intestinal delivery system was limited because if the porous shell structure. RESULTS Zein solution was pretreated with ultrasound and tannic acid (TA) cross‐linking. Composite oil‐core microcapsule (COM) with a compact shell structure was successfully prepared by using modified zein solution in the anti‐solvent process. Fourier‐transform infrared spectroscopy and circular dichroism analyses indicated that ultrasound and TA synergistically promote the conformational transition of zein from α‐helix to β‐sheet and enhance the hydrophobic interactions among protein chains. The above changes contribute to the strengthen of shell zein network. Correspondingly, COM presents superior encapsulation efficiency and environmental stability over the simple oil‐core microcapsule (SOM) prepared without the use of ultrasound and TA. Furthermore, antioxidant activity of β‐carotene was well retained during the encapsulation process. In vitro studies indicated that COM was more resistant to digestibility and acid‐induced swelling. More than 87% of β‐carotene could be released in the intestine in a sustainable way. The controllable release behavior thus promoted a significant increase in bioaccessibility of β‐carotene encapsulated in COM compared to SOM (85.9% versus 48.5%). CONCLUSION The COM generated here shows potential for bioaccessibility improvement of lipophilic ingredients. © 2022 Society of Chemical Industry.

Wei, Yun‐Shan↗

Characterization of a thermostable κ‐carrageenase from a hot spring bacterium and plant protection activity of the oligosaccharide enzymolysis product

Abstract Background Seaweed oligosaccharides are environmentally‐friendly natural products and their use for disease control in sustainable agriculture is extremely promising. Enzymatic digestion to prepare seaweed oligosaccharides has drawn considerable interest. However, the study of enzymatically degraded products of carrageenan is still in its infancy compared with that of other hydrocolloids such as agar and alginate. To prepare degraded carrageenan on a commercial scale, it is necessary to select superior producer bacterial strains to improve the yield and thermostability of carrageenases. Results The carrageenan‐degrading bacterium Bacillus sp. HT19 was isolated from sediment of a hot spring in Indonesia, and a κ‐carrageenase with high activity was purified from the culture supernatant. The purified enzyme, named Car19, had maximum activity (538 U mg −1 ) at 60 °C and pH 7.0. Notably, the enzyme retained >90% of its initial activity after incubation at 60 °C for 24 h. The Ca 2+ obviously improved the thermostability of Car19 at 70 °C. The K m and V max values of purified Car19 were 0.061 mg mL −1 and 115.13 U mg −1 , respectively, with κ‐carrageenan as substrate. Thin‐layer chromatography and electrospray ionization mass‐spectrometry analysis of hydrolysates indicated that the enzyme exolytically depolymerized κ‐carrageenan to neo‐carrabiose. The hydrolysate enhanced the resistance of cucumber to cucumber mosaic virus and increased the activity of antioxidant enzymes in infected plants. Conclusion To our knowledge, Car19 is the most thermostable κ‐carrageenase reported so far. Its high optimal reaction temperature and thermostability, and unitary hydrolysate constituent, makes Car19 a promising candidate for the preparation of carrageenan oligosaccharides with plant protection activity. © 2018 Society of Chemical Industry

Li, Jiang↗

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

16S rRNA↗

Plastic glut down a microbial gut

Enzymes sequestered from microbes have demonstrated the ability to readily digest amorphous regions of polyethylene terephthalate (PET) in ambient conditions. Although nascent, enzymatic depolymerization can soon vie for commercialization and provide monomeric feedstock at rates comparable to petrochemical feedstock for repolymerization, achieving the coveted goal of cradle-to-cradle recycling. © 2022 The Authors. Polymer International published by John Wiley & Sons Ltd on behalf of Society of Industrial Chemistry.

59 BASIC BIOLOGICAL SCIENCES↗

BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon

Abstract Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2 , an ortholog of the wheat TaGT43‐4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon . Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced in BdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon .

36 MATERIALS SCIENCE↗

A sorghum NAC gene is associated with variation in biomass properties and yield potential

Abstract Sorghum bicolor is a C 4 grass widely cultivated for grain, forage, sugar, and biomass. The sorghum Dry Stalk (D) locus controls a qualitative difference between juicy green (dd) and dry white (D‐) stalks and midribs, and co‐localizes with a quantitative trait locus for sugar yield. Here, we apply fine‐mapping and genome‐wide association study ( GWAS ) to identify a candidate gene underlying D , and use nearly isogenic lines (NILs) to characterize the transcriptional, compositional, and agronomic effects of variation at the D locus. The D locus was fine‐mapped to a 36 kb interval containing four genes. One of these genes is a NAC transcription factor that contains a stop codon in the NAC domain in the recessive (dd) parent. Allelic variation at D affects grain yield, sugar yield, and biomass composition in NILs. Green midrib ( dd) NIL s show reductions in lignin in stalk tissue and produce higher sugar and grain yields under well‐watered field conditions. Increased yield potential in dd NIL s is associated with increased stalk mass and moisture, higher biomass digestibility, and an extended period of grain filling. Transcriptome profiling of midrib tissue at the 4–6 leaf stages, when NIL s first become phenotypically distinct, reveals that dd NIL s have increased expression of a miniature zinc finger ( MIF ) gene. MIF genes dimerize with and suppress zinc finger homeodomain ( ZF ‐ HD ) transcription factors, and a ZF ‐ HD gene is associated with midrib color variation in a GWAS analysis across 1,694 diverse sorghum inbreds. A premature stop codon in a NAC gene is the most likely candidate polymorphism underlying the sorghum D locus. More detailed understanding of the sorghum D locus could help improve agronomic potential in cereals.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of soybean proteins from a single cell type: The root hair

Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.

Environmental Molecular Sciences Laboratory↗

Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies

Characterization of geomicrobial protein expression provides information necessary to better understand the unique biological pathways that occur within soil microbial communities and the role they play in regulating atmospheric CO2 levels and the Earth’s climate. A significant challenge in studying soil microbial proteins is their initial dissociation from the complex mixture of particles found in natural soil. Due to bias of the most robust cells, the removal of intact bacterial cells limits the characterization of the complete representation of a microbial community. However, in-situ lysis of bacterial cells leads to the expulsion of proteins to the soil surface, which can lead to potentially high levels of adsorption due to the physicochemical properties of both the protein and the soil. We investigated various compounds for their ability to block protein adsorption soil sites prior to in-situ lysis of bacterial cells, as well as their compatibility with both tryptic digestion and mass spectrometric analysis. The treatments were tested by adding lysed Escherichia coli proteins to representative treated and untreated soil samples. The results show that it is possible to significantly increase protein identifications through blockage of binding sites on a variety of soil textures; use of an optimized desorption buffer further increases the number of identifications.

block protein adsorption↗

Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Here, proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

59 BASIC BIOLOGICAL SCIENCES↗

Dissecting the structural heterogeneity of proteins by native mass spectrometry

Abstract A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

59 BASIC BIOLOGICAL SCIENCES↗

Integrated structural model of the palladin–actin complex using XL ‐ MS , docking, NMR , and SAXS

Abstract Palladin is an actin‐binding protein that accelerates actin polymerization and is linked to the metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin‐like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F‐actin. Evidence that palladin binds to the sides of actin filaments to facilitate branching is supported by our previous study showing that palladin was able to compensate for Arp2/3 in the formation of Listeria actin comet tails. Here, we used chemical crosslinking to covalently link palladin and F‐actin residues based on spatial proximity. Samples were then enzymatically digested, separated by liquid chromatography, and analyzed by tandem mass spectrometry. Peptides containing the crosslinks and specific residues involved were then identified for input to the HADDOCK docking server to model the most likely binding conformation. Small‐angle x‐ray scattering was used to provide further insight into palladin flexibility and the binding interface, and NMR spectra identified potential interactions between palladin's Ig domains. Our final structural model of the F‐actin:palladin complex revealed how palladin interacts with and stabilizes F‐actin at the interface between two actin monomers. Three actin residues that were identified in this study also appear commonly in the actin‐binding interface with other proteins such as myotilin, myosin, and tropomodulin. An accurate structural representation of the complex between palladin and actin extends our understanding of palladin's role in promoting cancer metastasis through the regulation of actin dynamics.

Sargent, Rachel [Department of Chemistry and Bioch↗

Fukushima Daiichi reactor source term attribution using cesium isotope ratios from contaminated environmental samples

Source term attribution of environmental contamination following the Fukushima Daiichi Nuclear Power Plant (FDNPP) disaster is complicated by a large number of possible similar emission source terms (e.g. FDNPP reactor cores 1–3 and spent fuel ponds 1–4). Cesium isotopic analyses can be utilized to discriminate between environmental contamination from different FDNPP source terms and, if samples are sufficiently temporally resolved, potentially provide insights into the extent of reactor core damage at a given time. In this work, rice, soil, mushroom, and soybean samples taken 100–250 km from the FDNPP site were dissolved using microwave digestion. Radiocesium was extracted and purified using two sequential ammonium molybdophosphate-polyacrylonitrile columns, following which 135 Cs/ 137 Cs isotope ratios were measured using thermal ionization mass spectrometry (TIMS). Results were compared with data reported previously from locations to the northwest of FDNPP and 30 km to the south of FDNPP. 135 Cs/ 137 Cs isotope ratios from samples 100–250 km to the southwest of the FDNPP site show a consistent value of 0.376 ± 0.008. 135 Cs/ 137 Cs versus 134 Cs/ 137 Cs correlation plots suggest that radiocesium to the southwest is derived from a mixture of FDNPP reactor cores 1, 2, and 3. Conclusions from the cesium isotopic data are in agreement with those derived independently based upon the event chronology combined with meteorological conditions at the time of the disaster. In conclusion, cesium isotopic analyses provide a powerful tool for source term discrimination of environmental radiocesium contamination at the FDNPP site. For higher precision source term attribution and forensic determination of the FDNPP core conditions based upon cesium, analyses of a larger number of samples from locations to the north and south of the FDNPP site (particularly time-resolved air filter samples) are needed. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.

59 BASIC BIOLOGICAL SCIENCES↗