Tau filaments with the Alzheimer fold in cases with MAPT mutations V337M and R406W.

Frontotemporal dementia (FTD) and Alzheimer's disease are the most common forms of early-onset dementia. Dominantly inherited mutations in MAPT , the microtubule-associated protein tau gene, cause FTD and parkinsonism linked to chromosome 17 (FTDP-17). Individuals with FTDP-17 develop abundant filamentous tau inclusions in brain cells. Here we used electron cryo-microscopy to determine the structures of tau filaments from the brains of individuals with MAPT mutations V337M and R406W. Both mutations gave rise to tau filaments with the Alzheimer fold, which consisted of paired helical filaments in all V337M and R406W cases and of straight filaments in two V337M cases. We also identified a new assembly of the Alzheimer fold into triple tau filaments in a V337M case. Filaments assembled from recombinant tau(297-391) with mutation V337M had the Alzheimer fold and showed an increased rate of assembly.

Cryo-EM structures of Aß40 filaments from the leptomeninges of individuals with Alzheimer's disease and cerebral amyloid angiopathy.

We used electron cryo-microscopy (cryo-EM) to determine the structures of Aß40 filaments from the leptomeninges of individuals with Alzheimer's disease and cerebral amyloid angiopathy. In agreement with previously reported structures, which were solved to a resolution of 4.4 Å, we found three types of filaments. However, our new structures, solved to a resolution of 2.4 Å, revealed differences in the sequence assignment that redefine the fold of Aß40 peptides and their interactions. Filaments are made of pairs of protofilaments, the ordered core of which comprises D1-G38. The different filament types comprise one, two or three protofilament pairs. In each pair, residues H14-G37 of both protofilaments adopt an extended conformation and pack against each other in an anti-parallel fashion, held together by hydrophobic interactions and hydrogen bonds between main chains and side chains. Residues D1-H13 fold back on the adjacent parts of their own chains through both polar and non-polar interactions. There are also several additional densities of unknown identity. Sarkosyl extraction and aqueous extraction gave the same structures. By cryo-EM, parenchymal deposits of Aß42 and blood vessel deposits of Aß40 have distinct structures, supporting the view that Alzheimer's disease and cerebral amyloid angiopathy are different Aß proteinopathies.

Cryptic kin discrimination during communal lactation in mice favours cooperation between relatives.

Breeding females can cooperate by rearing their offspring communally, sharing synergistic benefits of offspring care but risking exploitation by partners. In lactating mammals, communal rearing occurs mostly among close relatives. Inclusive fitness theory predicts enhanced cooperation between related partners and greater willingness to compensate for any partner under-investment, while females are less likely to bias investment towards own offspring. We use a dual isotopic tracer approach to track individual milk allocation when familiar pairs of sisters or unrelated house mice reared offspring communally. Closely related pairs show lower energy demand and pups experience better access to non-maternal milk. Lactational investment is more skewed between sister partners but females pay greater energetic costs per own offspring reared with an unrelated partner. The choice of close kin as cooperative partners is strongly favoured by these direct as well as indirect benefits, providing a driver to maintain female kin groups for communal breeding.

Operation of a TCA cycle subnetwork in the mammalian nucleus.

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110ß Signaling.

The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110ß signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood-brain barrier (BBB) limits drugs' brain penetration. We aimed to discover EGFR/PI3Kp110ß pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure-activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110ß, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110ß, and two dual inhibitors) decreased cell viability by 40-99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets' downregulation and increased apoptosis (15-85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110ß inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

Is there a Role for Antenatal Corticosteroids in Term Infants before Elective Cesarean Section?

OBJECTIVE: Cesarean section (CS) delivery, especially without previous labor, is associated with worse neonatal respiratory outcomes. Some studies comparing neonatal outcomes between term infants exposed and not exposed to antenatal corticosteroids (ACS) before elective CS revealed that ACS appears to decrease the risk of respiratory distress syndrome (RDS), transient tachypnea of the neonate (TTN), admission to the neonatal intensive care unit (NICU), and the length of stay in the NICU. METHODS: The present retrospective cohort study aimed to compare neonatal outcomes in infants born trough term elective CS exposed and not exposed to ACS. Outcomes included neonatal morbidity at birth, neonatal respiratory morbidity, and general neonatal morbidity. Maternal demographic characteristics and obstetric data were analyzed as possible confounders. RESULTS: A total of 334 newborns met the inclusion criteria. One third of the population study (n = 129; 38.6%) received ACS. The present study found that the likelihood for RDS (odds ratio [OR] = 1.250; 95% confidence interval [CI]: 0.454-3.442), transient TTN (OR = 1.,623; 95%CI: 0.556-4.739), and NIUC admission (OR = 2.155; 95%CI: 0.474-9.788) was higher in the ACS exposed group, although with no statistical significance. When adjusting for gestational age and arterial hypertension, the likelihood for RDS (OR = 0,732; 95%CI: 0.240-2.232), TTN (OR = 0.959; 95%CI: 0.297-3.091), and NIUC admission (OR = 0,852; 95%CI: 0.161-4.520) become lower in the ACS exposed group. CONCLUSION: Our findings highlight the known association between CS-related respiratory morbidity and gestational age, supporting recent guidelines that advocate postponing elective CSs until 39 weeks of gestational age.

Is there a role for antenatal corticosteroids in term infants before elective cesarean section?

Abstract Objective Cesarean section (CS) delivery, especially without previous labor, is associated with worse neonatal respiratory outcomes. Some studies comparing neonatal outcomes between term infants exposed and not exposed to antenatal corticosteroids (ACS) before elective CS revealed that ACS appears to decrease the risk of respiratory distress syndrome (RDS), transient tachypnea of the neonate (TTN), admission to the neonatal intensive care unit (NICU), and the length of stay in the NICU. Methods The present retrospective cohort study aimed to compare neonatal outcomes in infants born trough term elective CS exposed and not exposed to ACS. Outcomes included neonatal morbidity at birth, neonatal respiratory morbidity, and general neonatal morbidity. Maternal demographic characteristics and obstetric data were analyzed as possible confounders. Results A total of 334 newborns met the inclusion criteria. One third of the population study (n=129; 38.6%) received ACS. The present study found that the likelihood for RDS (odds ratio [OR]=1.250; 95% confidence interval [CI]: 0.454-3.442), transient TTN (OR=1.,623; 95%CI: 0.556-4.739), and NIUC admission (OR=2.155; 95%CI: 0.474-9.788) was higher in the ACS exposed group, although with no statistical significance. When adjusting for gestational age and arterial hypertension, the likelihood for RDS (OR=0,732; 95%CI: 0.240-2.232), TTN (OR=0.959; 95%CI: 0.297--3.091), and NIUC admission (OR=0,852; 95%CI: 0.161-4.520) become lower in the ACS exposed group. Conclusion Our findings highlight the known association between CS-related respiratory morbidity and gestational age, supporting recent guidelines that advocate postponing elective CSs until 39 weeks of gestational age.

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.

Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.

Social status and ejaculate composition in the house mouse.

Sperm competition theory predicts that males should tailor ejaculates according to their social status. Here, we test this in a model vertebrate, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our analyses reveal that both sperm production and the composition of proteins found in seminal vesicle secretions differ according to social status. Dominant males invested more in ejaculate production overall. Their epididymides contained more sperm than those of subordinate or control males, despite similar testes size between the groups. Dominant males also had larger seminal vesicle glands than subordinate or control males, despite similar body size. However, the seminal vesicle secretions of subordinate males had a significantly higher protein concentration than those of dominant males. Moreover, detailed proteomic analysis revealed subtle but consistent differences in the composition of secreted seminal vesicle proteins according to social status, involving multiple proteins of potential functional significance in sperm competition. These findings have significant implications for understanding the dynamics and outcome of sperm competition, and highlight the importance of social status as a factor influencing both sperm and seminal fluid investment strategies. This article is part of the theme issue 'Fifty years of sperm competition'.

[Metabolic Bone Disease of Prematurity in Very Low Birthweight Infants: Retrospective Observational Study]. / Doença Metabólica Óssea da Prematuridade em Recém-Nascidos de Muito Baixo Peso: Estudo Observacional Retrospetivo.

INTRODUCTION: Metabolic bone disease of prematurity consists in a decrease of bone matrix mineral content, in comparison with the level expected for gestational age. Screening of this condition is based on serum alkaline phosphatase and phosphate levels. The aim of this study is to evaluate the prevalence of metabolic bone disease of prematurity, to assess the aspects associated with a higher risk of this disease and to describe the growth of newborns with birth weight below 1500 g and metabolic bone disease of prematurity. MATERIAL AND METHODS: Observational, retrospective, multicenter and descriptive study in three neonatal intensive care units in Portugal, from May 1st 2016 to April 30th 2017. A convenience sample of very low birthweight newborns was obtained. Demographic, clinical, and laboratory variables were described in newborns with and without metabolic bone disease of prematurity. RESULTS: A total of 53 newborns were included in this study: 30 males, 16 with gestational age ≤ 28 weeks. Five cases of metabolic bone disease of prematurity were diagnosed. In this group, the majority of patients was male and presented a lower gestational age and birth weight, in comparison with the group without metabolic bone disease of prematurity. The average duration of parenteral nutrition was higher in newborns with metabolic bone disease of prematurity and the calcium/phosphate ratio was lower than the recommended values. Growth was similar in both groups. No patient with metabolic bone disease of prematurity underwent physical rehabilitation. DISCUSSION: The prevalence of metabolic bone disease of prematurity was 9.43%, which is lower than what is described in the literature. However, only 50% of newborns completed the screening according to the recommendations. The main risk factors identified concur with the literature. CONCLUSION: Metabolic bone disease of prematurity is a frequent but underdiagnosed comorbidity in very low birthweight newborns. It is essential to screen newborns at risk for this condition, using biochemical markers, as well as structure nutritional interventions and physical stimulation in order to avoid short and long-term consequences of this disease.

Introdução: A doença metabólica óssea da prematuridade consiste numa diminuição da matriz óssea, relativamente ao nível esperado para a idade gestacional. O rastreio baseia-se no doseamento sérico da fosfatase alcalina e fósforo. O objetivo deste estudo é avaliar a prevalência da doença metabólica óssea da prematuridade, analisar os aspetos associados a maior risco para esta doença e descrever o crescimento estaturo-ponderal dos recém-nascidos com peso ao nascer inferior a 1500 g, com doença metabólica óssea da prematuridade. Material e Métodos: Estudo multicêntrico, retrospetivo, observacional e descritivo em três unidades de apoio perinatal diferenciado, entre 1 de maio de 2016 e 30 de abril de 2017; foi obtida uma amostra de conveniência de recém-nascidos com muito baixo peso ao nascer. Descrevem-se as variáveis demográficas, clínicas e laboratoriais dos recém-nascidos com e sem doença metabólica óssea da prematuridade. Resultados: Neste estudo foram incluídos 53 recém-nascidos: 30 do sexo masculino, 16 com idade gestacional ≤ 28 semanas. Foram diagnosticados cinco casos de doença metabólica óssea da prematuridade. Neste grupo, a maioria dos doentes era do sexo masculino e apresentavam idade gestacional e peso ao nascer inferior aos do grupo sem doença metabólica óssea da prematuridade. A duração média de nutrição parentérica foi superior nos recém-nascidos com doença metabólica óssea da prematuridade e a relação cálcio/fósforo utilizada foi inferior às recomendações nacionais. A evolução estaturo-ponderal foi semelhante nos recém-nascidos com e sem doença. Nenhum doente com doença metabólica óssea da prematuridade teve intervenção por medicina física e reabilitação. Discussão: A prevalência de doença metabólica óssea da prematuridade foi de 9,43%, valor inferior ao descrito na literatura. Contudo, apenas 50% dos recém-nascidos cumpriram o rastreio de acordo com as recomendações. Os principais fatores de risco identificados estão de acordo com a literatura. Conclusão: A doença metabólica óssea da prematuridade é uma comorbilidade frequente nos recém-nascidos de muito baixo peso, mas encontra-se subdiagnosticada. É fundamental rastrear os recém-nascidos em risco para esta patologia, utilizando marcadores bioquímicos, assim como estruturar intervenções nutricionais e estimulação física para evitar as consequências da doença a curto e longo prazo.

Skin and coeliac disease, a lot to think about: a case series.

Coeliac disease (CD) is an autoimmune disease, characterised by a permanent sensitivity to gluten. It is being progressively recognised as a multisystemic disease, with multiple extraintestinal manifestations. Skin conditions (eg, dermatitis herpetiformis) are an example of its manifestations; however, its underlying mechanisms are still not well understood. This article presents three cases of uncommon skin conditions in patients with a history of CD. Two of them concern linear IgA bullous dermatosis and erythema nodosum, which have been described in the literature as having potential associations with CD, though only a few cases were reported. The third case corresponds to pityriasis lichenoides-a rare lymphoproliferative disorder of unknown aetiology-, which has no correlation with CD in the literature reviewed. The authors aim to draw attention to the possibility of CD as a potential predisposing factor for the occurrence of these skin diseases.

High resolution parallel reaction monitoring with electron transfer dissociation for middle-down proteomics.

In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.

Recent proteomic advances in developmental, regeneration, and cancer governing signaling pathways.

Embryonic development, adult tissue repair, and cancer share a number of common regulating pathways. The basic processes that govern the events that induce mesenchymal properties in epithelial cells-a process known as epithelial-mesenchymal transition-are central for embryonic development, and can be resumed in adults either during wound healing or tissue regeneration. A misregulation of these pathways is involved in pathological situations, such as tissue fibrosis and cancer. Proteomic approaches have emerged as promising tools to better understand the signaling pathways that govern these complex biological processes. This review focuses on the recent proteomic-based contributions to better understand the modulation of transforming growth factor-beta (TGF-ß), wingless-type MMTV integration site family (Wnt), Notch and Receptor tyrosine kinase (RTK) signaling pathways. New advances include the description of new protein interactions, the formation of new protein complexes or the description on how some PTMs are regulating these pathways. Understanding protein interactions and the tempo-spatial modulation of these pathways might lead us to interesting research quests in cancer, embryonic development or even on improving adult tissue regeneration capabilities.

Proteolytic events are relevant cellular responses during nervous system regeneration of the starfish Marthasterias glacialis.

The molecular pathways that trigger the amazing intrinsic regenerative ability of echinoderm nervous system are still unknown. In order to approach this subject, a 2D-DIGE proteomic strategy was used, to screen proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. A total of 528 proteins showed significant variations during radial nerve cord regeneration in both soluble and membrane protein-enriched fractions. Several functional classes of proteins known to be involved in axon regeneration events in other model organisms, such as chordates, were identified for the first time in the regenerating echinoderm nervous system. Unexpectedly, most of the identified proteins presented a molecular mass either higher or lower than expected. Such results suggest a functional modulation through protein post-translational modifications, such as proteolysis. Among these are proteins involved in cytoskeleton and microtubule regulators, axon guidance molecules and growth cone modulators, protein de novo synthesis machinery, RNA binding and transport, transcription factors, kinases, lipid signaling effectors and proteins with neuroprotective functions. In summary, the impact of proteolysis during regeneration events is here shown, although requiring further studies to detail on the mechanisms involving this post-transcriptional event on nervous system regeneration. BIOLOGICAL SIGNIFICANCE: The nervous systems of some organisms present a complete inability of neurons to regrow across a lesion site, which is the case of the adult mammalian central nervous system (CNS). Expanding our knowledge on how other animals regenerate their nervous system offers great potential for groundbreaking biomedical applications towards the enhancement of mammalian CNS regeneration. In order to approach this subject, a 2D-DIGE proteomic strategy was used for the first time, to screen the proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. We strongly believe in the relevance of our results and have clear evidences that this work constitutes a solid basis for new research on starfish regenerating nerve cord. We also believe this work will have a significant impact not only on the general scientific community as we present here an alternative animal model to neurobiology, but also on the scientific community that works with echinoderms or closely related marine invertebrates, which are constantly searching for specific protein markers of several tissues, thus constituting an important advance towards the improvement of large scale protein information of unsequenced, but yet not less important organisms.

A possible approach for gel-based proteomic studies in recalcitrant woody plants.

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.

Understanding regeneration through proteomics.

Regeneration is a complex cellular process that, rather than simply forming a scar following injury, the animal forms a new functional tissue. Regeneration is a widespread process among metazoa, although not uniformly. Planaria, starfish, and some worms can regenerate most of their body, whereas many other species can only regenerate parts of specific tissues or fail to accomplish a functional regrowth, as is the case of mammals CNS. Research in regenerative medicine will possibly culminate in the regeneration of organs/tissues originally not prone to this process. Despite the complexity of the interactions and regulatory systems involved, the variety of tissues and organs these cells differentiate into has so far impaired the success of direct transplantation to restore damaged tissues. For this reason, a study, at the molecular level of the regeneration mechanisms developed by different animal models is likely to provide answers to why these processes are not readily activated in mammals. Proteomic-based approaches are being recognized as extremely useful to study of regeneration events, also because there is a relevant contribution of posttranscriptional processes that involve frequently the occurrence of a broad range of PTMs. The present review focuses on the significant knowledge brought up by proteomics in diverse aspects of regeneration research on different animal models, tissues, and organs.