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The burgeoning human population has resulted in an augmented demand for raw materials and energy sources, which in turn has led to a deleterious environmental impact marked by elevated greenhouse gas (GHG) emissions, acidification of water bodies, and escalating global temperatures. Therefore, it is imperative that modern society develop sustainable technologies to avert future environmental degradation and generate alternative bioproduct-producing technologies. A promising approach to tackling this challenge involves utilizing natural microbial consortia or designing synthetic communities of microorganisms as a foundation to develop diverse and sustainable applications for bioproduct production, wastewater treatment, GHG emission reduction, energy crisis alleviation, and soil fertility enhancement. Microalgae, which are photosynthetic microorganisms that inhabit aquatic environments and exhibit a high capacity for CO 2 fixation, are particularly appealing in this context. They can convert light energy and atmospheric CO 2 or industrial flue gases into valuable biomass and organic chemicals, thereby contributing to GHG emission reduction. To date, most microalgae cultivation studies have focused on monoculture systems. However, maintaining a microalgae monoculture system can be challenging due to contamination by other microorganisms (e.g., yeasts, fungi, bacteria, and other microalgae species), which can lead to low productivity, culture collapse, and low-quality biomass. Co-culture systems, which produce robust microorganism consortia or communities, present a compelling strategy for addressing contamination problems. In recent years, research and development of innovative co-cultivation techniques have substantially increased. Nevertheless, many microalgae co-culturing technologies remain in the developmental phase and have yet to be scaled and commercialized. Accordingly, this review presents a thorough literature review of research conducted in the last few decades, exploring the advantages and disadvantages of microalgae co-cultivation systems that involve microalgae-bacteria, microalgae-fungi, and microalgae-microalgae/algae systems. The manuscript also addresses diverse uses of co-culture systems, and growing methods, and includes one of the most exciting research areas in co-culturing systems, which are omic studies that elucidate different interaction mechanisms among microbial communities. Finally, the manuscript discusses the economic viability, future challenges, and prospects of microalgal co-cultivation methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Naseema Rasheed, Pourbakhtiar, Mehdizadeh Allaf, Baharlooeian, Rafiei, Alishah Aratboni, Morones-Ramirez and Winck.)

Brazil would benefit from a long-term strategy for science and innovation that improves the standing of both science and scientists in the country., Competing Interests: GC, JM, RC, FW No competing interests declared, (© 2023, Crestana et al.)

The metabolism of the model microalgae Chlamydomonas reinhardtii under nitrogen deprivation is of special interest due to its resulting increment of triacylglycerols (TAGs), that can be applied in biotechnological applications. However, this same condition impairs cell growth, which may limit the microalgae's large applications. Several studies have identified significant physiological and molecular changes that occur during the transition from an abundant to a low or absent nitrogen supply, explaining in detail the differences in the proteome, metabolome and transcriptome of the cells that may be responsible for and responsive to this condition. However, there are still some intriguing questions that reside in the core of the regulation of these cellular responses that make this process even more interesting and complex. In this scenario, we reviewed the main metabolic pathways that are involved in the response, mining and exploring, through a reanalysis of omics data from previously published datasets, the commonalities among the responses and unraveling unexplained or non-explored mechanisms of the possible regulatory aspects of the response. Proteomics, metabolomics and transcriptomics data were reanalysed using a common strategy, and an in silico gene promoter motif analysis was performed. Together, these results identified and suggested a strong association between the metabolism of amino acids, especially arginine, glutamate and ornithine pathways to the production of TAGs, via the de novo synthesis of lipids. Furthermore, our analysis and data mining indicate that signalling cascades orchestrated with the indirect participation of phosphorylation, nitrosylation and peroxidation events may be essential to the process. The amino acid pathways and the amount of arginine and ornithine available in the cells, at least transiently during nitrogen deprivation, may be in the core of the post-transcriptional, metabolic regulation of this complex phenomenon. Their further exploration is important to the discovery of novel advances in the understanding of microalgae lipids' production.

Decellularized ECMs have been used as biological scaffolds for tissue repair due to their tissue-specific biochemical and mechanical composition, poorly simulated by other materials. It is used as patches and powders, and it could be further processed via enzymatic digestion under acidic conditions using pepsin. However, part of the bioactivity is lost during the digestion process due to protein denaturation. Here, stepwise digestion was developed to prepare a competent biomaterial for osteogenesis from three different ECM sources. In addition, three different proteases were compared to evaluate the most effective digestion protocol for specific cellular processes. GAGs and peptide quantification showed that the stepwise method yielded a higher concentration of bioactive residues. Circular dichroism analysis also showed that the stepwise approach preserved the secondary structures better. The protein profiles of the digested ECMs were analyzed, and it was found to be highly diverse and tissue-specific. The digestion of ECM from pericardium produced peptides originated from 94 different proteins, followed by 48 proteins in ECM from tendon and 35 proteins in ECM from bone. In addition, digested products from pericardium ECM yielded increased proliferation and differentiation of bone marrow mesenchymal stem cells to mature osteoblasts., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Gabriela M. Monteiro-Lobato et al.)

Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell- and SCC-9 cell-derived EVs. A multi-omics integration identified 11 'hub proteins' significantly decreased at the metastatic site compared with primary tumor-derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven 'hub proteins' in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteome analysis of model and non-model plants is a genuine scientific field in expansion. Several technological advances have contributed to the implementation of different proteomics approaches for qualitative and quantitative analysis of the dynamics of cellular responses at the protein level. The design of time-resolved experiments and the emergent use of multiplexed proteome analysis using chemical or isotopic and isobaric labeling strategies as well as label-free approaches are generating a vast amount of proteomics data that is going to be essential for analysis of protein posttranslational modifications and implementation of systems biology approaches. Through the target proteomics analysis, especially the ones that combine the untargeted methods, we should expect an improvement in the completeness of the identification of proteome and reveal nuances of regulatory cellular mechanisms related to plant development and responses to environmental stresses. Both genomic sequencing and proteomic advancements in the last decades coupled to integrative data analysis are enriching biological information that was once confined to model plants. Therewith, predictions of a changing environment places proteomics as an especially useful tool for crops performance., (© 2021. Springer Nature Switzerland AG.)

How the complexity of biological systems can be understood is currently limited by the amount of biological information we have available to be incorporate in the vastitude of possibilities that could represent how a biological organism function. This point of view is, of course, alive under the paradigm that describes a living thing as a whole that could never be interpreted as to the sole understanding of its separated parts.If we are going to achieve the knowledge to understand all the complex relations between the molecules, pathways, organelles, cells, organs, phenotypes, and environments is unknown. However, that is exactly what moves us toward digging the most profound nature of relationships present in the living organisms.During the last 20 years, a big workforce was dedicated to the development of techniques, instruments, and scientific approaches that guided a whole new generation of scientists into the universe of omics approaches. The implementation of technological advances in several omics applications, such as transcriptomics, proteomics, and metabolomics, has brought to light the information that nowadays reshape our previous thinking on specific aspects of plant sciences, including growth, development, organ communication, chromatin states, and metabolism, not to mention the underpinning role of regulatory mechanisms that in many cases are essentially the basis for the phenotypical expression of a biological phenomenon and plants adaptation to their environment.In this chapter, some of the original concepts of complex systems theory were briefly discussed, and examples of omics approaches that are contributing to uncovering emergent characteristics of plants are presented and discussed. The combination of several experimental and computational or mathematical approaches indicated that there is room for improvements and novel discoveries. However, the level of complexity of biological systems seems to require and demand us to unify efforts toward the integration of the large omics datasets already available and the ones to come. This unification may represent the necessary breakthrough to the achievement of the understanding of complex phenomena. To do so, the inclusion of systems biology thinking into the training of undergraduate and graduate students of plant sciences and related areas seems to be also a contribution that is necessary to be organized and implemented in a worldwide scale., (© 2021. Springer Nature Switzerland AG.)

Background: The increasing amounts of genomics data have helped in the understanding of the molecular dynamics of complex systems such as plant and animal diseases. However, transcriptional regulation, although playing a central role in the decision-making process of cellular systems, is still poorly understood. In this study, we linked expression data with mathematical models to infer gene regulatory networks (GRN). We present a simple yet effective method to estimate transcription factors' GRNs from transcriptional data., Method: We defined interactions between pairs of genes (edges in the GRN) as the partial mutual information between these genes that takes into account time and possible lags in time from one gene in relation to another. We call this method Gene Regulatory Networks on Transfer Entropy (GRNTE) and it corresponds to Granger causality for Gaussian variables in an autoregressive model. To evaluate the reconstruction accuracy of our method, we generated several sub-networks from the GRN of the eukaryotic yeast model, Saccharomyces cerevisae. Then, we applied this method using experimental data of the plant pathogen Phytophthora infestans. We evaluated the transcriptional expression levels of 48 transcription factors of P. infestans during its interaction with one moderately resistant and one susceptible cultivar of yellow potato (Solanum tuberosum group Phureja), using RT-qPCR. With these data, we reconstructed the regulatory network of P. infestans during its interaction with these hosts., Results: We first evaluated the performance of our method, based on the transfer entropy (GRNTE), on eukaryotic datasets from the GRNs of the yeast S. cerevisae. Results suggest that GRNTE is comparable with the state-of-the-art methods when the parameters for edge detection are properly tuned. In the case of P. infestans, most of the genes considered in this study, showed a significant change in expression from the onset of the interaction (0 h post inoculum - hpi) to the later time-points post inoculation. Hierarchical clustering of the expression data discriminated two distinct periods during the infection: from 12 to 36 hpi and from 48 to 72 hpi for both the moderately resistant and susceptible cultivars. These distinct periods could be associated with two phases of the life cycle of the pathogen when infecting the host plant: the biotrophic and necrotrophic phases., Conclusions: Here we presented an algorithmic solution to the problem of network reconstruction in time series data. This analytical perspective makes use of the dynamic nature of time series data as it relates to intrinsically dynamic processes such as transcription regulation, were multiple elements of the cell (e.g., transcription factors) act simultaneously and change over time. We applied the algorithm to study the regulatory network of P. infestans during its interaction with two hosts which differ in their level of resistance to the pathogen. Although the gene expression analysis did not show differences between the two hosts, the results of the GRN analyses evidenced rewiring of the genes' interactions according to the resistance level of the host. This suggests that different regulatory processes are activated in response to different environmental cues. Applications of our methodology showed that it could reliably predict where to place edges in the transcriptional networks and sub-networks. The experimental approach used here can help provide insights on the biological role of these interactions on complex processes such as pathogenicity. The code used is available at https://github.com/jccastrog/GRNTE under GNU general public license 3.0.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

The increase in atmospheric CO 2 due to anthropogenic activities is generating climate change, which has resulted in a subsequent rise in global temperatures with severe environmental impacts. Biological mitigation has been considered as an alternative for environmental remediation and reduction of greenhouse gases in the atmosphere. In fact, the use of easily adapted photosynthetic organisms able to fix CO 2 with low-cost operation is revealing its high potential for industry. Among those organism, the algae Chlamydomonas reinhardtii have gain special attention as a model organism for studying CO 2 fixation, biomass accumulation and bioenergy production upon exposure to several environmental conditions. In the present study, we studied the Chlamydomonas response to different CO 2 levels by comparing metabolomics and transcriptomics data with the predicted results from our new-improved genomic-scale metabolic model. For this, we used in silico methods at steady dynamic state varying the levels of CO 2 . Our main goal was to improve our capacity for predicting metabolic routes involved in biomass accumulation. The improved genomic-scale metabolic model presented in this study was shown to be phenotypically accurate, predictive, and a significant improvement over previously reported models. Our model consists of 3726 reactions and 2436 metabolites, and lacks any thermodynamically infeasible cycles. It was shown to be highly sensitive to environmental changes under both steady-state and dynamic conditions. As additional constraints, our dynamic model involved kinetic parameters associated with substrate consumption at different growth conditions (i.e., low CO 2 -heterotrophic and high CO 2 -mixotrophic). Our results suggest that cells growing at high CO 2 (i.e., photoautotrophic and mixotrophic conditions) have an increased capability for biomass production. In addition, we have observed that ATP production also seems to be an important limiting factor for growth under the conditions tested. Our experimental data (metabolomics and transcriptomics) and the results predicted by our model clearly suggest a differential behavior between low CO 2 -heterotrophic and high CO 2 -mixotrophic growth conditions. The data presented in the current study contributes to better dissect the biological response of C. reinhardtii, as a dynamic entity, to environmental and genetic changes. These findings are of great interest given the biotechnological potential of this microalga for CO 2 fixation, biomass accumulation, and bioenergy production., Competing Interests: The authors declare there are no competing interests.

EEF1D (eukaryotic translation elongation factor 1δ) is a subunit of the elongation factor 1 complex of proteins that mediates the elongation process during protein synthesis via enzymatic delivery of aminoacyl-tRNAs to the ribosome. Although the functions of EEF1D in the translation process are recognized, EEF1D expression was found to be unbalanced in tumours. In the present study, we demonstrate the overexpression of EEF1D in OSCC (oral squamous cell carcinoma), and revealed that EEF1D and protein interaction partners promote the activation of cyclin D1 and vimentin proteins. EEF1D knockdown in OSCC reduced cell proliferation and induced EMT (epithelial-mesenchymal transition) phenotypes, including cell invasion. Taken together, these results define EEF1D as a critical inducer of OSCC proliferation and EMT., (© 2016 Authors; published by Portland Press Limited.)

The development of microalgae sustainable applications needs better understanding of microalgae biology. Moreover, how cells coordinate their metabolism toward biomass accumulation is not fully understood. In this present study, flux balance analysis (FBA) was performed to identify sensitive metabolic pathways of Chlamydomonas reinhardtii under varied CO2 inputs. The metabolic network model of Chlamydomonas was updated based on the genome annotation data and sensitivity analysis revealed CO2 sensitive reactions. Biological experiments were performed with cells cultivated at 0.04% (air), 2.5, 5, 8, and 10% CO2 concentration under controlled conditions and cell growth profiles and biomass content were measured. Pigments, lipids, proteins, and starch were further quantified for the reference low (0.04%) and high (10%) CO2 conditions. The expression level of candidate genes of sensitive reactions was measured and validated by quantitative real time PCR. The sensitive analysis revealed mitochondrial compartment as the major affected by changes on the CO2 concentrations and glycolysis/gluconeogenesis, glyoxylate, and dicarboxylate metabolism among the affected metabolic pathways. Genes coding for glycerate kinase (GLYK), glycine cleavage system, H-protein (GCSH), NAD-dependent malate dehydrogenase (MDH3), low-CO2 inducible protein A (LCIA), carbonic anhydrase 5 (CAH5), E1 component, alpha subunit (PDC3), dual function alcohol dehydrogenase/acetaldehyde dehydrogenase (ADH1), and phosphoglucomutase (GPM2), were defined, among other genes, as sensitive nodes in the metabolic network simulations. These genes were experimentally responsive to the changes in the carbon fluxes in the system. We performed metabolomics analysis using mass spectrometry validating the modulation of carbon dioxide responsive pathways and metabolites. The changes on CO2 levels mostly affected the metabolism of amino acids found in the photorespiration pathway. Our updated metabolic network was compared to previous model and it showed more consistent results once considering the experimental data. Possible roles of the sensitive pathways in the biomass metabolism are discussed.

Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.

The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

Proteomics experiments often generate a vast amount of data. However, the simple identification and quantification of proteins from a cell proteome or subproteome is not sufficient for the full understanding of complex mechanisms occurring in the biological systems. Therefore, the functional annotation analysis of protein datasets using bioinformatics tools is essential for interpreting the results of high-throughput proteomics. Although large-scale proteomics data have rapidly increased, the biological interpretation of these results remains as a challenging task. Here we reviewed basic concepts and different programs that are commonly used in proteomics data functional annotation, emphasizing the main strategies focused in the use of gene ontology annotations. Furthermore, we explored the characteristics of some tools developed for functional annotation analysis, concerning the ease of use and typical caveats on ontology annotations. The utility and variations between different tools were assessed through the comparison of the resulting outputs generated for an example of proteomics dataset., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species., (© 2013 Institute of Botany, Chinese Academy of Sciences.)

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R 2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance., (© 2014 American Society of Plant Biologists. All rights reserved.)

Unlabelled: Oral cancer disease represents a significant fraction of all human cancer types and its poor early diagnosis contributes to reduced individual survival rate. The identification of proteins modulated in tumorigenic cells and its post-translational modifications may improve our understanding of tumor development in epithelial cells. We have analyzed the phosphoproteome of tumorigenic (SCC-9) and non-tumorigenic (HaCaT) cell lines using MS-based approach in order to identify phosphopeptides with differing patterns of modifications and/or abundance. Our results revealed the identity of 4,206 protein phosphorylation sites with sixty-two sites showing to be significantly modulated between the two cell lines. The phosphoproteome data showed an overrepresentation of proteins with a possible role in nuclear regulatory functions. Pathway analysis was further performed on the phosphoproteome dataset and differences and commonalities of the functional pathways present in tumorigenic and non-tumorigenic cells were identified. Phosphopeptides that belong to the proteins lamina-associated polypeptide 2 isoform alpha and serine-arginine repetitive matrix protein 2 were identified with differential abundance and they appear as promising tumor-related phosphopeptides. These two proteins may be related to the structural alterations generally found in the nucleus of tumorigenic cells. The identification of phosphorylation sites in tumorigenic cells may contribute to disclose novel signaling mechanisms associated with OSCC., Significance: Oral Squamous Cell Carcinoma (OSCC) is an important cancer disease affecting thousands of people worldwide. Many cellular processes related to the development of oral cancer remain unknown; however, the studies performed in vitro with cancer cells have contributed to guide more specific research which may be further performed by using in vivo approaches or clinical samples. To our knowledge, only few studies have been published showing the results of phosphoproteome profiling of squamous cell carcinoma models, and many signaling proteins must be identified and functionally characterized in order to increase the knowledge available about the complexity of the signaling networks responsible for oral cancer development and its progression. Furthermore, our knowledge regarding proteins exclusive or very low abundant in cancer cells remains limited. A better understanding of the differences between signaling pathways present in epithelial cell lines may contribute to reveal the processes underlying the OSCC., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Understanding the processes and mechanisms of carbon acquisition and accumulation in microalgae is fundamental to enhance the cellular capabilities aimed to environmental and industrial applications. The "omics" approaches have greatly contributed to expanding the knowledge on these carbon-related cellular responses, reporting large data sets on microalgae transcriptome, proteome and metabolome. This review emphasizes the advances made on Chlamydomonas exploration; however, some knowledge acquired from studying this model organism, may be extrapolated to close algae species. The large data sets available for this organism revealed the identity of a vast range of genes and proteins which are integrating carbon-related mechanisms. Nevertheless, these data sets have also highlighted the need for integrative analysis in order to fully explore the information enclosed. Here, some of the main results from "omics" approaches which may contribute to the understanding of carbon acquisition and accumulation in Chlamydomonas were reviewed and possible applications were discussed., Biological Significance: A number of important publications in the field of "omics" technologies have been published reporting studies of the model green microalga Chlamydomonas reinhardtii and related to microalgal biomass production. However, there are only few attempts to integrate these data. Publications showing the results from "omics" approaches, such as transcriptome, metabolome and proteome, focused in the study of mechanisms of carbon acquisition and accumulation in microalgae were reviewed. This review contributes to highlight the knowledge recently generated on such "omics" studies and it discusses how these results may be important for the advance of applied sciences, such as microalgae biotechnology., (© 2013.)

The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1) and Lcr2 (Low-CO2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in Chlamydomonas.

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.

Nuclear proteins play a central role in regulating gene expression. Their identification is important for understanding how the nuclear repertoire changes over time under different conditions. Nuclear proteins are often underrepresented in proteomic studies due to the frequently low abundance of proteins involved in regulatory processes. So far, only few studies describing the nuclear proteome of plant species have been published. Recently, the genome sequence of the unicellular green alga Chlamydomonas reinhardtii has been obtained and annotated, allowing the development of further detailed studies for this organism. However, a detailed description of its nuclear proteome has not been reported so far. Here, we present an analysis of the nuclear proteome of the sequenced Chlamydomonas strain cc503. Using LC-MS/MS, we identified 672 proteins from nuclei isolates with a maximum 1% peptide spectrum false discovery rate. Besides well-known proteins (e.g. histones), transcription factors and other transcriptional regulators (e.g. tubby and HMG) were identified. The presence of protein motifs in nuclear proteins was investigated by computational tools, and specific over-represented protein motifs were identified. This study provides new insights into the complexity of the nuclear environment and reveals novel putative protein targets for further studies of nuclear mechanisms., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches., (© 2011 Phycological Society of America.)

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BArhoNA, Michaelis-Menten behavior with K(m) 2.3x10(-1)M and the V(max) 0.52x10(-1) nmoles rho-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause alpha- and beta-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Proteomic research has proved valuable for understanding the molecular mechanisms of biological processes, as well as in the search for biomarkers for a variety of diseases which lack a molecular diagnostic. While several new approaches are being developed, two-dimensional (2-DE) gel electrophoresis is still one of the most commonly used techniques, despite its many limitations. However, for biomarker research, 2-DE gel electrophoresis alone does not fulfill the necessary pre-requisites. If such a technique is utilized exclusively, a great part of a given proteome remains unseen. Therefore, very precise and sensitive techniques are needed. Here, we present a brief review of known methodologies that try to overcome the limitations of conventional proteome analysis as well as their respective advantages and limitations.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized D: -lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.

The fastidious bacterium Xylella fastidiosa is associated with important crop diseases worldwide. We have recently shown that X. fastidiosa is a peculiar organism having unusually low values of gene codon bias throughout its genome and, unexpectedly, in the group of the most abundant proteins. Here, we hypothesized that the lack of codon usage optimization in X. fastidiosa would incapacitate this organism to undergo quick and massive changes in protein expression as occurs in a classical stress response. Proteomic analysis of the response to heat stress in X. fastidiosa revealed that no changes in protein expression can be detected. Moreover, stress-inducible proteins identified in the closely related citrus pathogen Xanthomonas axonopodis pv citri were found to be constitutively expressed in X. fastidiosa. These proteins have extremely high codon bias values in the X. citri and other well-studied organisms, but low values in X. fastidiosa. Because biased codon usage is well known to correlate to the rate of protein synthesis, we speculate that the peculiar codon bias distribution in X. fastidiosa is related to the absence of a classical stress response, and, probably, alternative strategies for survival of X. fastidiosa under stressfull conditions.

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.

Xylella fastidiosa (Xf) is a fastidious bacterium that grows exclusively in the xylem of several important crop species, including grape and sweet orange (Citrus sinensis L. Osb.) causing Pierce disease and citrus variegated chlorosis (CVC), respectively. The aim of this work was to study the nitrogen metabolism of a highly susceptible variety of sweet orange cv. 'Pêra' (C. sinensis L. Osbeck) infected with Xf. Plants were artificially infected and maintained in the greenhouse until they have developed clear disease symptoms. The content of nitrogen compounds and enzymes of the nitrogen metabolism and proteases in the xylem sap and leaves of diseased (DP) and uninfected healthy (HP) plants was studied. The activity of nitrate reductase in leaves did not change in DP, however, the activity of glutamine synthetase was significantly higher in these leaves. Although amino acid concentration was slightly higher in the xylem sap of DP, the level dropped drastically in the leaves. The protein contents were lower in the sap and in leaves of DP. DP and HP showed the same amino acid profiles, but different proportions were observed among them, mainly for asparagine, glutamine, and arginine. The polyamine putrescine was found in high concentrations only in DP. Protease activity was higher in leaves of DP while, in the xylem sap, activity was detected only in DP. Bidimensional electrophoresis showed a marked change in the protein pattern in DP. Five differentially expressed proteins were identified (2 from HP and 3 from DP), but none showed similarity with the genomic (translated) and proteomic database of Xf, but do show similarity with the proteins thaumatin, mucin, peroxidase, ABC-transporter, and strictosidine synthase. These results showed that significant changes take place in the nitrogen metabolism of DP, probably as a response to the alterations in the absorption, assimilation and distribution of N in the plant.

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 mu-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I)-->Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35-45 degrees C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P<0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA2 was similar to another in the basic PLA2 of the crotoxin complex from C. durissus cascavella venom.

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).