Predicting the future of our oceans-Evaluating genomic forecasting approaches in marine species.

Climate change is restructuring biodiversity on multiple scales and there is a pressing need to understand the downstream ecological and genomic consequences of this change. Recent advancements in the field of eco-evolutionary genomics have sought to include evolutionary processes in forecasting species' responses to climate change (e.g., genomic offset), but to date, much of this work has focused on terrestrial species. Coastal and offshore species, and the fisheries they support, may be even more vulnerable to climate change than their terrestrial counterparts, warranting a critical appraisal of these approaches in marine systems. First, we synthesize knowledge about the genomic basis of adaptation in marine species, and then we discuss the few examples where genomic forecasting has been applied in marine systems. Next, we identify the key challenges in validating genomic offset estimates in marine species, and we advocate for the inclusion of historical sampling data and hindcasting in the validation phase. Lastly, we describe a workflow to guide marine managers in incorporating these predictions into the decision-making process.

Systemic therapies for salivary gland carcinomas: an overview of published clinical trials.

BACKGROUND: There is no consensus about effective systemic therapy for salivary gland carcinomas (sgcs). Our aim was summarized the clinical trials assessing the systemic therapies (ST) on sgcs. MATERIAL AND METHODS: Electronic searches were carried out through MEDLINE/pubmed, EMBASE, Scopus, Web of Science, and the Cochrane Library databases, and gray literature. RESULTS: Seventeen different drugs were evaluated, and the most frequent histological subtype was adenoid cystic carcinoma (n=195, 45.5%). Stable disease, observed in 11 ST, achieved the highest rate in adenoid cystic carcinoma treated with sunitinib. The highest complete (11.1%) and partial response (30.5%) rates were seen in androgen receptor-positive tumors treated with leuprorelin acetate. CONCLUSIONS: Despite all the advances in this field, there is yet no effective evidence-based regimen of ST, with all the clinical trials identified showing low rates of complete and partial responses. Further, translational studies are urgently required to characterize molecular targets and effective ST.

MutSα expression predicts a lower disease-free survival in malignant salivary gland tumors: an immunohistochemical study.

BACKGROUND: Appropriate DNA replication is vital to maintain cell integrity at the genomic level. Malfunction on DNA repair mechanisms can have implications related to tumor behavior. Our aim was to evaluate the expression of key complexes of the DNA mismatch-repair system MutSα (hMSH2-hMSH6) and MutSß (hMSH2-hMSH3) in a panel comprising the most common benign and malignant salivary gland tumors (SGT), and to determine their association with disease-free survival. MATERIAL AND METHODS: Ten cases of normal salivary gland (NSG) and 92 of SGT (54 benign and 38 malignant) were retrieved. Immunohistochemistry was performed for hMSH2, hMSH3, hMSH6. Scanned slides were digitally analyzed based on the percentage of positive cells with nuclear staining. Cases were further classified in MutSαhigh and MutSßhigh based on hMSH2-hMSH6 and hMSH3-hMSH6 expression, respectively. RESULTS: hMSH3 expression was lower in malignant SGT compared to NSG and benign cases. Adenoid cystic carcinoma (ACC) cases with perineural invasion presented a lower percentage of hMSH3 positive cells. hMSH6 was downregulated in both benign and malignant SGT compared to NSG. Malignant SGT cases with MutSαhigh expression had lower disease-free survival compared to MutSαlow cases. A 10.26-fold increased risk of presenting local recurrence was observed. CONCLUSIONS: Our findings suggest that a lack of hMSH3 protein function is associated with a more aggressive phenotype (malignancy and perineural invasion) and that MutSα overexpression predicts a poor clinical outcome in malignant SGT.

Establishment of a coastal fish in the Azores: recent colonisation or sudden expansion of an ancient relict population?

The processes and timescales associated with ocean-wide changes in the distribution of marine species have intrigued biologists since Darwin's earliest insights into biogeography. The Azores, a mid-Atlantic volcanic archipelago located >1000 km off the European continental shelf, offers ideal opportunities to investigate phylogeographic colonisation scenarios. The benthopelagic sparid fish known as the common two-banded seabream (Diplodus vulgaris) is now relatively common along the coastline of the Azores archipelago, but was virtually absent before the 1990 s. We employed a multiple genetic marker approach to test whether the successful establishment of the Azorean population derives from a recent colonisation from western continental/island populations or from the demographic explosion of an ancient relict population. Results from nuclear and mtDNA sequences show that all Atlantic and Mediterranean populations belong to the same phylogroup, though microsatellite data indicate significant genetic divergence between the Azorean sample and all other locations, as well as among Macaronesian, western Iberian and Mediterranean regions. The results from Approximate Bayesian Computation indicate that D. vulgaris has likely inhabited the Azores for ∼ 40 (95% confidence interval (CI): 5.5-83.6) to 52 (95% CI: 6.32-89.0) generations, corresponding to roughly 80-150 years, suggesting near-contemporary colonisation, followed by a more recent demographic expansion that could have been facilitated by changing climate conditions. Moreover, the lack of previous records of this species over the past century, together with the absence of lineage separation and the presence of relatively few private alleles, do not exclude the possibility of an even more recent colonisation event.

Single and double ionization of the camphor molecule excited around the C 1s edge.

RATIONALE: An interesting class of volatile compounds, the monoterpenes, is present in some plants although their functions are not yet fully understood. We have studied the interaction of the camphor molecule with monochromatic high-energy photons (synchrotron radiation) using time-of-flight mass spectrometry and coincidence techniques. METHODS: A commercial sample of S-camphor was admitted into the vacuum chamber, without purification, through an inlet system. Monochromatic light with energy around the C 1s edge was generated by the TGM beamline at the Brazilian Synchrotron Facility. A Wiley-McLaren mass spectrometer was used to characterize and detect the ions formed by the camphor photoionization. The data analysis was supported by energy calculations. RESULTS: Although the fragmentation patterns were basically the same at 270 eV and 330 eV, it was observed that above the C 1s edge the contribution to the spectrum from lower mass/charge fragment ions increased, pointing to a higher degree of dissociation of the molecule. Projections of the PEPIPICO spectra demonstrated the existence of unstable doubly charged species. The Gibbs free energy was calculated using the Møller-Plesset perturbation theory (MP2) for the neutral, singly and doubly excited camphor molecule. CONCLUSIONS: Our PEPIPICO spectrum clearly demonstrated the formation of doubly ionic dissociative species. From a slope analysis, we propose a secondary decay after a deferred charge separation mechanism in which, after a few steps, the camphor dication dissociates into C2 H3 (+) and C3 H5 (+) . This is the main relaxation route observed at 270 eV and 330 eV. The large energy difference between the mono and the dication (of the order of 258.2 kcal/mol) may explain the experimentally observed absence of stable dications in the spectra, because their formation is disadvantaged energetically.

Cyclin D1-induced proliferation is independent of beta-catenin in head and neck cancer.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) progression and metastasis have previously been associated with the activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) and Wnt signalling pathways, which lead to the activation of pro-proliferative genes, such as cyclin D1. The current study aims to investigate whether there is a crosstalk between these pathways in HNSCC and which pathway is more likely to regulate cyclin D1. MATERIAL AND METHODS: Two HNSCC and a control keratinocyte cell lines were treated with EGF and wortmannin to respectively activate and block the PI3K-Akt and Wnt pathways. Partial and total levels of cyclin D1, beta-catenin and Akt were evaluated by Western blotting and immunofluorescence. Twenty-four paraffin-embedded samples of human HNSCC, as well as normal oral mucosa biopsies, were also immunohistochemically evaluated for beta-catenin and cyclin D1 expression. RESULTS: Following both treatments, change in cyclin D1 protein was correlated with Akt levels only. Cytoplasmic staining for beta-catenin and loss of its membranous expression in the HNSCC invasive areas were found in 92% of the HNSCC biopsies. CONCLUSION: Taken together, we show that the change in cyclin D1 levels is more likely to be due to the EGFR-Akt pathway activation than due to beta-catenin nuclear translocation.

Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing.

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3K/Akt/mTOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3K/Akt/mTOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3K-PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds.

Comparative analysis between extra-short implants (≤6 mm) and 6 mm-longer implants: a meta-analysis of randomized controlled trial.

The goal of this systematic study was to compare the survival rate (SR), marginal bone loss (MBL) and clinical complications between extra-short implants (≤6 mm) and 6-mm-longer implants in randomized clinical trials. A systematic electronic and manual search was performed using the PubMed, Web of Science, Scopus and DOAJ databases. A meta-analysis was conducted to compare the SR and MBL between both groups. We have selected 17 studies out of 1016 articles for qualitative and quantitative analysis. The data from 956 patients and 1779 implants were used with an overall mean clinical follow-up of 3.88 years ranging from 1 to 8 years. Overall, the SR of extra-short implants (93.12%) was lower than the observed in 6-mm-longer implants (95.98%); however, there was no statistical significance on these findings (P > 0.10). MBL analysis showed that extra-short implants and the 6-mm-longer group presented an average of -0.71 and -0.92 mm after 1-year respectively. Three years follow-up showed MBL of -0.42 mm (≤6 mm) and -0.43 mm (>6 mm); 5 years follow-up showed an MBL of -0.69 mm (≤6 mm) and -0.46 mm (>6 mm); and after 8 years of follow-up, it was found an MBL of -1.58 mm (≤6 mm) and -2.46 mm (>6 mm). Within the limitation of this study, the results indicated that SR of extra-short implants was similar to 6-mm-longer implants. In contrast, MBL and the presence of clinical complications were observed at a lessened rate on extra-short implants.

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil.

AIMS: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. METHODS AND RESULTS: The antiviral activity was evaluated against human herpesvirus type 1 (HSV-1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV-WR) and dengue virus 2 (DENV-2) by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti-HSV-1 [EC(50) 142.8 ± 5.3 µg ml(-1); selectivity index (SI) 2.0] and anti-DENV-2 activity (EC(50) 9.8 ± 1.3 µg ml(-1) ; SI 1.5). The pectolinarin (1) isolated from LEE was less active against HSV-1 and DENV-2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV-1. A mixture of pectolinarin (1) and acacetin-7-O-rutinoside (2) was isolated from FEE and has presented anti-DENV-2 activity (EC(50) 11.1 ± 1.6 µg ml(-1) ; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. CONCLUSIONS: These data shows that D. elongata has antiviral activity mainly against HSV-1 and DENV-2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: Distictella elongata might be considered a promising source of anti-dengue fever phytochemicals.

Association or Causation? Exploring the Oral Microbiome and Cancer Links.

Several epidemiological investigations have found associations between poor oral health and different types of cancer, including colorectal, lung, pancreatic, and oral malignancies. The oral health parameters underlying these relationships include deficient oral hygiene, gingival bleeding, and bone and tooth loss. These parameters are related to periodontal diseases, which are directly and indirectly mediated by oral bacteria. Given the increased accessibility of microbial sequencing platforms, many recent studies have investigated the link between the oral microbiome and these cancers. Overall, it seems that oral dysbiotic states can contribute to tumorigenesis in the oral cavity as well as in distant body sites. Further, it appears that certain oral bacterial species can contribute to carcinogenesis, in particular, Fusobacterium nucleatum and Porphyromonas gingivalis, based on results from epidemiological as well as mechanistic studies. Yet, the strength of the findings from these investigations is hampered by the heterogeneity of the methods used to measure oral diseases, the treatment of confounding factors, the study design, the platforms employed for microbial analysis, and types of samples analyzed. Despite these limitations, there is an overall indication that the presence of oral dysbiosis that leads to oral diseases may directly and/or indirectly contribute to carcinogenesis. Proper methodological standardized approaches should be implemented in future epidemiological studies as well as in the mechanistic investigations carried out to explore these results.

Proctolaelaps bickleyi (Acari: Mesostigmata: Melicharidae): First Record of Its Association with Cotton Boll Weevil.

The objective was to register the occurrence of Proctolaelaps bickleyi (Bram) (Acari: Mesostigmata: Melicharidae) in association with adults of the Anthonomus grandis Boheman (Coleoptera: Curculionidae) and to describe aspects of its behavior. This is the first record of a mesostigmatid mite associated with the cotton boll weevil in the Americas. Beetles carrying the mites had lower mobility than usual. The stress caused by this mite attached to the A. grandis body can reduce mating and oviposition of this beetle.

Requirement of Rac1 distinguishes follicular from interfollicular epithelial stem cells.

Epithelial stem cells in the bulge region within the hair follicle maintain the cyclic hair growth, but whether these stem cells also contribute to the epidermal renewal remains unclear. Here, we observed that the conditional deletion of the Rac1 gene in the mouse skin, including the potential follicular and epidermal stem cell compartments, results in alopecia owing to defective hair development. Surprisingly, mice lacking the expression of this Rho GTPase do not display major alterations in the interfollicular skin. Furthermore, Rac1 excision from primary epithelial keratinocytes results in the inability to reconstitute hair follicles and sebaceous glands when grafted onto mice, but epithelial cells lacking Rac1 can nonetheless form a healthy epidermis. Together, these findings support the emerging view that the epidermis and the hair follicles are maintained by different epithelial stem cells, and provide evidence that the requirement for Rac1 function can distinguish these distinct stem cells populations.

Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake.

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Epigenetic Modifications of Histones in Periodontal Disease.

Periodontitis is a chronic infectious disease driven by dysbiosis, an imbalance between commensal bacteria and the host organism. Periodontitis is a leading cause of tooth loss in adults and occurs in about 50% of the US population. In addition to the clinical challenges associated with treating periodontitis, the progression and chronic nature of this disease seriously affect human health. Emerging evidence suggests that periodontitis is associated with mechanisms beyond bacteria-induced protein and tissue degradation. Here, we hypothesize that bacteria are able to induce epigenetic modifications in oral epithelial cells mediated by histone modifications. In this study, we found that dysbiosis in vivo led to epigenetic modifications, including acetylation of histones and downregulation of DNA methyltransferase 1. In addition, in vitro exposure of oral epithelial cells to lipopolysaccharides resulted in histone modifications, activation of transcriptional coactivators, such as p300/CBP, and accumulation of nuclear factor-κB (NF-κB). Given that oral epithelial cells are the first line of defense for the periodontium against bacteria, we also evaluated whether activation of pathogen recognition receptors induced histone modifications. We found that activation of the Toll-like receptors 1, 2, and 4 and the nucleotide-binding oligomerization domain protein 1 induced histone acetylation in oral epithelial cells. Our findings corroborate the emerging concept that epigenetic modifications play a role in the development of periodontitis.

Ca2+ acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by phenylarsine oxide.

Mitochondrial permeability transition (MPT) induced by the thiol cross-linker phenylarsine oxide (PhAsO) in Ca(2+)-depleted mitochondria incubated in the presence of ruthenium red, an inhibitor of the Ca2+ uniporter, is stimulated by the addition of extramitochondrial Ca2+. The presence of extramitochondrial Ca2+ stimulates the reaction of mitochondrial membrane protein thiol groups with PhAsO. Both Ca(2+)-induced increase in mitochondrial membrane permeabilization and protein thiol group reaction with PhAsO are dependent on time (5-10 min to be complete) and the concentration of Ca2+ (1-25 microM). Mitochondrial permeabilization induced by PhAsO (15 microM) and extramitochondrial Ca2+ is inhibited by ADP, cyclosporin A, dibucaine and Mg2+, while mitochondrial permeabilization induced by high concentrations of PhAsO (60 microM) in the absence of Ca2+ is inhibited only by ADP and cyclosporin A. These results suggest that dibucaine and Mg2+ can inhibit mitochondrial permeabilization by antagonizing the effect of Ca2+ on the mitochondrial membrane. Once mitochondrial permeabilization induced by 15 microM PhAsO and extramitochondrial Ca2+ has already occurred, the addition of the Ca2+ chelator EGTA restores mitochondrial membrane potential (MPT pore closure), suggesting that the presence of Ca2+ is essential for the maintenance of the permeability of the mitochondrial membrane to protons (MPT pore opening). In conclusion, the results presented indicate that low Ca2+ concentrations acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by PhAsO, due to increased accessibility of protein thiol groups to the reaction with PhAsO and increased probability of MPT pore opening.

Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition.

The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid.

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive oxygen species (ROS), at concentrations as low as 50-100 microM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porphyria patients. Removal of Ca2+ (10 microM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swelling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca2+. Addition of MgCl2 at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca2+ efflux. This indicates that Mg2+ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.

Ca(2+)-dependent permeabilization of the inner mitochondrial membrane by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955-19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

Oxidative damage of mitochondria induced by 5-aminolevulinic acid: role of Ca2+ and membrane protein thiols.

Reactive oxygen species (ROS) generated by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation have been shown to damage the inner membrane of isolated rat liver mitochondria by a Ca(2+)-dependent mechanism. The present work describes experiments indicating that this damage can be prevented, but not completely reversed by the additions of catalase, ADP, cyclosporin A and dithiothreitol, as judged by the extent of delta psi regeneration by the injured mitochondria. In contrast, the addition of EGTA, which removes free Ca2+ and, possibly, Fe2+ present both in the intra- and extramitochondrial compartments, causes a prompt and complete regeneration of delta psi, even after long periods of mitochondrial incubations in the presence of ALA. This reversibility suggests that protein alterations such as protein thiol cross-linkings, evidenced by SDS-polyacrylamide gel electrophoresis, are the main cause of increased membrane permeability promoted by ALA oxidation. The inhibition of protein aggregation and fast regeneration of delta psi promoted by EGTA suggest that the binding of Ca2+ to some membrane proteins plays a crucial role in the mechanism of both protein polymerization (pore assembly) and pore opening. The implication of these results with the molecular pathology of acute intermittent porphyria is also discussed.