•Hydrothermal liquefaction is an effective biocrude production method for macroalgae.•Biocrude yield is highly influenced by biomass organic carbon content.•Assessment of biocrude productivities is ...an effective species selection tool.•Marine macroalgae have higher biocrude productivities due to higher growth rates.
Six species of marine and freshwater green macroalgae were cultivated in outdoor tanks and subsequently converted to biocrude through hydrothermal liquefaction (HTL) in a batch reactor. The influence of the biochemical composition of biomass on biocrude yield and composition was assessed. The freshwater macroalgae Oedogonium afforded the highest biocrude yield of all six species at 26.2%, dry weight (dw). Derbesia (19.7%dw) produced the highest biocrude yield for the marine species followed by Ulva (18.7%dw). In contrast to significantly different yields across species, the biocrudes elemental profiles were remarkably similar with higher heating values of 33–34MJkg−1. Biocrude productivity was highest for marine Derbesia (2.4gm−2d−1) and Ulva (2.1gm−2d−1), and for freshwater Oedogonium (1.3gm−2d−1). These species were therefore identified as suitable feedstocks for scale-up and further HTL studies based on biocrude productivity, as a function of biomass productivity and the yield of biomass conversion to biocrude.
In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances ...in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation.
Multiple testing corrections are a useful tool for restricting the FDR, but can be blunt in the context of low power, as we demonstrate by a series of simple simulations. Unfortunately, in proteomics ...experiments low power can be common, driven by proteomics‐specific issues like small effects due to ratio compression, and few replicates due to reagent high cost, instrument time availability and other issues; in such situations, most multiple testing corrections methods, if used with conventional thresholds, will fail to detect any true positives even when many exist. In this low power, medium scale situation, other methods such as effect size considerations or peptide‐level calculations may be a more effective option, even if they do not offer the same theoretical guarantee of a low FDR. Thus, we aim to highlight in this article that proteomics presents some specific challenges to the standard multiple testing corrections methods, which should be employed as a useful tool but not be regarded as a required rubber stamp.
Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and ...reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15-100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.
Food safety of staple crops such as rice is of global concern and is at the top of the policy agenda worldwide. Abiotic stresses are one of the main limitations to optimizing yields for ...sustainability, food security and food safety. We analyzed proteome changes in
cv. Nipponbare in response to five adverse abiotic treatments, including three levels of drought (mild, moderate, and severe), soil salinization, and non-optimal temperatures. All treatments had modest, negative effects on plant growth, enabling us to identify proteins that were common to all stresses, or unique to one. More than 75% of the total of differentially abundant proteins in response to abiotic stresses were specific to individual stresses, while fewer than 5% of stress-induced proteins were shared across all abiotic constraints. Stress-specific and non-specific stress-responsive proteins identified were categorized in terms of core biological processes, molecular functions, and cellular localization.
Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses ...to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96 h of cold stress at 12–14°C. The use of both label‐free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label‐free approach identified 236 cold‐responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress.
The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until ...12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.
In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.
Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.
Oryza meridionalis Ng. is a wild relative of Oryza sativa L. found throughout northern Australia where temperatures regularly exceed 35 °C in the monsoon growing season. Heat tolerance in O. ...meridionalis was established by comparing leaf elongation and photosynthetic rates at 45 °C with plants maintained at 27 °C. By comparison with O. sativa ssp. japonica cv. Amaroo, O. meridionalis was heat tolerant. Elongation rates of the third leaf of O. meridionalis declined by 47% over 24 h at 45 °C compared with a 91% decrease for O. sativa. Net photosynthesis was significantly higher in O. sativa at 27 °C whereas the two species had the same assimilation rates at 45 °C. The leaf proteome and expression levels of individual heat-responsive genes provided insight into the heat response of O. meridionalis. After 24 h of heat exposure, many enzymes involved in the Calvin Cycle were more abundant, while mRNA of their genes generally decreased. Ferredoxin-NADP(H) oxidoreductase, a key enzyme in photosynthetic electron transport had both reduced abundance and gene expression, suggesting light reactions were highly susceptible to heat stress. Rubisco activase was strongly up-regulated after 24 h of heat, with the large isoform having the largest relative increase in protein abundance and a significant increase in gene expression. The protective proteins Cpn60, Hsp90, and Hsp70 all increased in both protein abundance and gene expression. A thiamine biosynthesis protein (THI1), previously shown to act protectively against stress, increased in abundance during heat, even as thiamine levels fell in O. meridionalis.
Glaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma ...and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer's disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases.
Root system architecture (RSA) is critical for improving nutrient and water uptake and maintaining crop yield under both optimal and drought conditions. The meristematic zone of root tips is ideal ...for studying RSA, because of its high mitotic rate. miRNAs are important post-transcriptional regulators and play a crucial role in plant response to drought stress. To decipher the changes in miRNA expression patterns under drought stress, sequencing of small RNAs in the meristematic root tips of the highly tolerant genotypes Azucena and the susceptible IR64 was performed. Prediction of target genes of differentially expressed miRNAs (DEMs) in drought-stressed Azucena compared with normal conditions revealed that these genes mainly encode aquaporin, AP2, DELLA protein, ERF, and OsIAA18-auxin responsive. Most of these genes are involved in the regulation of response to stress, meristem growth, ethylene-activated signaling pathway, post-transcriptional regulation, and lipid metabolism. Whereas, the predicted target genes in IR64 under the same conditions were mainly involved in controlling carbohydrate biosynthesis, lignin catabolism, primary metabolic processes, cell death, and RNAi. This result shows that IR64 promotes thickening of cell walls to cope with stress, while lateral root system expansion and root length are used as a strategy to cope with drought stress in Azucena. Azucena-specific down-regulation of miR164-NAC5, miR1857-MADX-box5, miR1861-EXP1, and miR169-NF-YA may be the main reason for root elongation, root system development, and stress adaptation of this genotype. Moreover, miR528 and miR398, which serve as hub regulators, and target redox-related enzymes such as SOD and peroxidase (POX) to control cellular homeostasis in response to drought stress, were down-regulated in Azucena. Furthermore, our histochemical assay also confirmed the fact that the cell wall thickness of the root tips was increased in the drought-sensitive IR64 for insulation purposes to cope with stress. However, this may substantially reduce its ability to extend roots to obtain water from deeper soil layers. Our results shed light on drought-related miRNA regulatory networks as well as important miRNAs that have the potential to further improve drought tolerance in rice.
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