RESUMO
Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3â months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.
Assuntos
Envelhecimento/fisiologia , Autofagia , Células Epiteliais/citologia , Água Doce , Hydra/fisiologia , Células-Tronco/citologia , Animais , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Temperatura Baixa , Epiderme/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Gametogênese/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hydra/efeitos dos fármacos , Hydra/genética , Imageamento Tridimensional , Fenótipo , Inibidores de Proteassoma/farmacologia , Sirolimo/farmacologia , Células-Tronco/efeitos dos fármacos , Análise de SobrevidaRESUMO
Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked.
Assuntos
Ciclo Celular/fisiologia , Hydra/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Animais , Ciclo Celular/genética , Divisão Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes cdc , Hydra/citologia , Hydra/efeitos dos fármacos , Hydra/genética , Hidroxiureia/farmacologia , Nocodazol/farmacologia , Regeneração/efeitos dos fármacos , Regeneração/genética , Fase S , TranscriptomaRESUMO
The impact of injury-induced immune responses on animal regenerative processes is highly variable, positive or negative depending on the context. This likely reflects the complexity of the innate immune system that behaves as a sentinel in the transition from injury to regeneration. Early-branching invertebrates with high regenerative potential as Hydra provide a unique framework to dissect how injury-induced immune responses impact regeneration. A series of early cellular events likely require an efficient immune response after amputation, as antimicrobial defence, epithelial cell stretching for wound closure, migration of interstitial progenitors toward the wound, cell death, phagocytosis of cell debris, or reconstruction of the extracellular matrix. The analysis of the injury-induced transcriptomic modulations of 2636 genes annotated as immune genes in Hydra identified 43 genes showing an immediate/early pulse regulation in all regenerative contexts examined. These regulations point to an enhanced cytoprotection via ROS signaling (Nrf, C/EBP, p62/SQSMT1-l2), TNFR and TLR signaling (TNFR16-like, TRAF2l, TRAF5l, jun, fos-related, SIK2, ATF1/CREB, LRRC28, LRRC40, LRRK2), proteasomal activity (p62/SQSMT1-l1, Ced6/Gulf, NEDD8-conjugating enzyme Ubc12), stress proteins (CRYAB1, CRYAB2, HSP16.2, DnaJB9, HSP90a1), all potentially regulating NF-κB activity. Other genes encoding immune-annotated proteins such as NPYR4, GTPases, Swap70, the antiproliferative BTG1, enzymes involved in lipid metabolism (5-lipoxygenase, ACSF4), secreted clotting factors, secreted peptidases are also pulse regulated upon bisection. By contrast, metalloproteinases and antimicrobial peptide genes largely follow a context-dependent regulation, whereas the protease inhibitor α2macroglobulin gene exhibits a sustained up-regulation. Hence a complex immune response to injury is linked to wound healing and regeneration in Hydra.
Assuntos
Hydra/imunologia , Hydra/fisiologia , Imunidade Inata , Animais , Plantas/imunologia , Espécies Reativas de Oxigênio/metabolismo , Regeneração , Transdução de SinaisRESUMO
BACKGROUND: Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. RESULTS: To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. CONCLUSIONS: We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.
Assuntos
Genoma , Hydra/genética , Transcriptoma , Animais , Hibridização Genômica Comparativa , Hydra/classificação , Fases de Leitura Aberta , Filogenia , Análise de Sequência de RNARESUMO
After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.
Assuntos
Apoptose/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hydra/metabolismo , Hydra/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Hydra/genética , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/genéticaRESUMO
A variety of polymer nanoparticles (NP) are under development for imaging and therapeutic use. However, little is known about their behavior. This study examined pharmacokinetics, distribution and elimination of stable polyacrylamide (PAA) nanoparticles (~31 nm average diameter). PAA NPs and polyethylene glycol-coated PAA NPs were injected into the tail veins of healthy male rats. Blood, tissues and excreta were collected at times ranging from 5 min to 120 h and their radioactive content was quantified. A mathematical model was then applied to analyze the distribution dynamics of both NPs. Elimination from the blood could be accounted for by a quick but finite relocation to the major organs (about 20%, 0.6 to 1.3h half-lives), and a slower distribution to the carcass (about 70%, 35 to 43 h half-lives). Excreted urinary levels correlated with blood concentrations. Combined cumulative urinary and fecal output accounted for less than 6% of the dose at 120 h. Compared to five other polymeric nanoparticles, the studied particles are at the highest half-lives and Area Under the Curve (4000 to 5000%-h). These two parameters decrease by three orders of magnitude when nanoparticle size increases from the 30 nm range up to 250 nm. For similar sizes, pegylated nanoparticles are more persistent in the blood than non-pegylated ones, but this difference is much smaller in the 30 nm and relatively high dose range than above 100 nm. Persistence of PAA NPs is not associated with acute toxicity signs as measured by typical serum markers of inflammation and cellular damage.
Assuntos
Resinas Acrílicas/farmacocinética , Portadores de Fármacos/farmacocinética , Nanopartículas , Polietilenoglicóis/farmacocinética , Resinas Acrílicas/administração & dosagem , Resinas Acrílicas/toxicidade , Animais , Área Sob a Curva , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/toxicidade , Meia-Vida , Injeções Intravenosas , Masculino , Modelos Teóricos , Tamanho da Partícula , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/toxicidade , Ratos , Distribuição Tecidual , Testes de Toxicidade AgudaRESUMO
OBJECTIVES: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. CONCLUSIONS: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.
Assuntos
COVID-19/diagnóstico , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/normas , Artefatos , COVID-19/virologia , Genoma Viral , Técnicas de Genotipagem/métodos , Guias como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Viral , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma/métodos , Fluxo de TrabalhoRESUMO
Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when ß-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra, acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/ß-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via ß-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/ß-catenin signaling, a function conserved across eumetazoan evolution.
Assuntos
Hydra/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Wnt3/genética , beta Catenina/genética , Animais , Evolução Biológica , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/crescimento & desenvolvimento , Cabeça/fisiologia , Hydra/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Planárias/genética , Interferência de RNA , Regeneração/fisiologia , Transdução de Sinais , Proteína Wnt3/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismoRESUMO
CONTEXT: For the general population, the dominant source of exposure to dioxin-like compounds is food. As part of the University of Michigan Dioxin Exposure Study (UMDES), we measured selected polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) in serum of 946 subjects who were a representative sample of the general population in five Michigan counties. CASE PRESENTATION: The total toxic equivalency (TEQ; based on 2005 World Health Organization toxic equivalency factors) of serum from the index case was 211 ppt on a lipid-adjusted basis, which was the highest value observed in the UMDES study population. This subject had no apparent opportunity for exposure to dioxins, except that she had lived on property with soil contaminated with dioxins for almost 30 years, and had been a ceramics hobbyist for > 30 years. Soil from her property and clay that she used for ceramics were both contaminated with dioxins, but the congener patterns differed. DISCUSSION: The congener patterns in this subject's serum, soil, and ceramic clay suggest strongly that the dioxin contamination in clay and not soil was the dominant source of dioxin contamination in her serum. RELEVANCE TO PUBLIC HEALTH PRACTICE: It appears that ceramic clay, in particular the process of firing clay with unvented kilns, can be a significant nonfood and nonindustrial source of human exposure to dioxins among ceramics hobbyists. The extent of human exposure from ceramic clay is unclear, but it may be widespread. Further work is needed to more precisely characterize the routes of exposure.
Assuntos
Silicatos de Alumínio , Dioxinas/toxicidade , Exposição Ambiental , Idoso , Argila , Dioxinas/sangue , Feminino , HumanosRESUMO
An essential dimension of 3D regeneration in adult animals is developmental, with the formation of organizers from somatic tissues. These organizers produce signals that recruit surrounding cells and drive the restoration of the missing structures (organs, appendages, body parts). However, even in animals with a high regenerative potential, this developmental potential is not sufficient to achieve regeneration as homeostatic conditions at the time of injury need to be "pro-regenerative". In Hydra, we identified four distinct homeostatic properties that provide a pro-regenerative framework and we discuss here how these non-developmental properties impact regeneration. First, both the epithelial and the interstitial-derived cells are highly plastic along the animal body, a plasticity that offers several routes to achieve regeneration. Second, the abundant stocks of continuously self-renewing adult stem cells form a constitutive pro-blastema in the central body column, readily activated upon bisection. Third, the autophagy machinery in epithelial cells guarantees a high level of fitness and adaptation to detrimental environmental conditions, as evidenced by the loss of regeneration in animals where autophagy is dysfunctional. Fourth, the extracellular matrix, named mesoglea in Hydra, provides a dynamically-patterned environment where the molecular and mechanical signals induced by injury get translated into a regenerative process. We claim that these homeostatic pro-regenerative features contribute to define the high regenerative potential of adult Hydra.
Assuntos
Homeostase/fisiologia , Hydra/fisiologia , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Hydra/citologia , Modelos Biológicos , Células-Tronco/citologiaRESUMO
The growth and patterning of anatomical structures from specific cellular fields in developing organisms relies on organizing centers that instruct surrounding cells to modify their behavior, namely migration, proliferation, and differentiation. We discuss here how organizers can form in adult organisms, a process of utmost interest for regenerative medicine. Animals like Hydra and planarians, which maintain their shape and fitness thanks to a highly dynamic homeostasis, offer a useful paradigm to study adult organizers in steady-state conditions. Beside the homeostatic context, these model systems also offer the possibility to study how organizers form de novo from somatic adult tissues. Both extracellular matrix remodeling and caspase activation play a key role in this transition, acting as promoters of organizer formation in the vicinity of the wound. Their respective roles and the crosstalk between them just start to be deciphered.
Assuntos
Diferenciação Celular , Hydra/crescimento & desenvolvimento , Organizadores Embrionários/metabolismo , Planárias/crescimento & desenvolvimento , Regeneração/fisiologia , Adulto , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hydra/metabolismo , Planárias/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
Phenotypic traits derive from the selective recruitment of genetic materials over macroevolutionary times, and protein-coding genes constitute an essential component of these materials. We took advantage of the recent production of genomic scale data from sponges and cnidarians, sister groups from eumetazoans and bilaterians, respectively, to date the emergence of human proteins and to infer the timing of acquisition of novel traits through metazoan evolution. Comparing the proteomes of 23 eukaryotes, we find that 33% human proteins have an ortholog in nonmetazoan species. This premetazoan proteome associates with 43% of all annotated human biological processes. Subsequently, four major waves of innovations can be inferred in the last common ancestors of eumetazoans, bilaterians, euteleostomi (bony vertebrates), and hominidae, largely specific to each epoch, whereas early branching deuterostome and chordate phyla show very few innovations. Interestingly, groups of proteins that act together in their modern human functions often originated concomitantly, although the corresponding human phenotypes frequently emerged later. For example, the three cnidarians Acropora, Nematostella, and Hydra express a highly similar protein inventory, and their protein innovations can be affiliated either to traits shared by all eumetazoans (gut differentiation, neurogenesis); or to bilaterian traits present in only some cnidarians (eyes, striated muscle); or to traits not identified yet in this phylum (mesodermal layer, endocrine glands). The variable correspondence between phenotypes predicted from protein enrichments and observed phenotypes suggests that a parallel mechanism repeatedly produce similar phenotypes, thanks to novel regulatory events that independently tie preexisting conserved genetic modules.
Assuntos
Sequência Conservada/genética , Evolução Molecular , Hominidae/genética , Filogenia , Animais , Cnidários/genética , Genômica , Hominidae/classificação , Humanos , Proteínas/classificação , Proteínas/genética , ProteomaRESUMO
OBJECTIVE: In this study we reviewed the half-life data in the literature for the 29 dioxin, furan, and polychlorinated biphenyl congeners named in the World Health Organization toxic equivalency factor scheme, with the aim of providing a reference value for the half-life of each congener in the human body and a method of half-life estimation that accounts for an individual's personal characteristics. DATA SOURCES AND EXTRACTION: We compared data from >30 studies containing congener-specific elimination rates. Half-life data were extracted and compiled into a summary table. We then created a subset of these data based on defined exclusionary criteria. DATA SYNTHESIS: We defined values for each congener that approximate the half-life in an infant and in an adult. A linear interpolation of these values was used to examine the relationship between half-life and age, percent body fat, and absolute body fat. We developed predictive equations based on these relationships and adjustments for individual characteristics. CONCLUSIONS: The half-life of dioxins in the body can be predicted using a linear relationship with age adjusted for body fat, smoking, and breast-feeding. Data suggest an alternative method based on a linear relationship between half-life and total body fat, but this approach requires further testing and validation with individual measurements.
Assuntos
Dioxinas/química , Furanos/química , Bifenilos Policlorados/química , Adulto , Fatores Etários , Composição Corporal , Aleitamento Materno , Dioxinas/farmacocinética , Feminino , Furanos/farmacocinética , Meia-Vida , Humanos , Lactente , Masculino , Modelos Biológicos , Bifenilos Policlorados/farmacocinética , Valores de Referência , FumarRESUMO
Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.
Assuntos
Apoptose/fisiologia , Hydra , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Clorometilcetonas de Aminoácidos/metabolismo , Animais , Proliferação de Células , Hydra/anatomia & histologia , Hydra/fisiologia , Marcação In Situ das Extremidades Cortadas , Interferência de RNA , Fase S/fisiologia , Proteínas Wnt/genética , Proteína Wnt3 , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.