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The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64 839 bp revealed 21 protein-coding genes and several hypothetical open reading frames with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related to the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in noncoding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.

The filamentous fungus Neurospora crassa is a popular model organism used in a wide range of biochemical and genetic studies and vastly used in mitochondrial research. Despite the relevance of mitochondria in N. crassa biology, no method for quantification of mitochondrial DNA (mtDNA) is currently available. Quantitative real-time PCR (qPCR) is a powerful tool, with a wide range of applications, and has been used for the quantification of nucleic acids in humans and a few other species. Here we present a new qPCR assay for relative quantification of N. crassa mtDNA. Three sets of qPCR primers targeting different regions of the mitochondrial genome were tested for mtDNA quantification. The qPCR was successfully validated in N. crassa strains from different geographical locations, representing the vast genetic diversity of this species, and knockout mutant strains. Moreover the assay proved to be efficient in templates with varied amounts of mitochondria, obtained through different DNA extraction methods. The qPCR performed well in all tested samples revealing a higher amount of mtDNA than nuclear DNA in all cases. This technique will facilitate the characterization of mtDNA of N. crassa in future studies and can be used as a tool to validate methods of mitochondria isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: The standardization of quantitative real-time PCR (qPCR) techniques is essential to enable and facilitate future comparisons. Neurospora crassa is a model organism with a lot of potential in different fields of study. Here we use N. crassa to develop and establish an assay to quantify mitochondrial DNA using qPCR. We tested strains with different geographical background and our data demonstrated the usefulness of this assay to quantify mitochondrial DNA in N. crassa. This technique can be useful in a wide variety of applications and in different types of studies.

Plasma biochemical analysis remains one of the established ways of monitoring captive marine mammal health. More recently, complementary plasma lipidomic analysis has proven to be a valid tool in disease diagnosis and prevention, with the potential to validate and complement common biochemical analysis, providing a more integrative approach. In this study, we thoroughly characterized the plasma polar lipid content of Tursiops truncatus, the most common cetacean species held under human care. Our results showed that phosphatidylcholine, lysophosphatidylcholine, and sphingomyelins (CerPCho) are the most represented phospholipid classes in T. truncatus plasma. Palmitic, oleic, and stearic acids are the major fatty acid (FA) present esterified to the plasma polar lipids of this species, although some n-3 species are also remarkably present, namely eicosapentaenoic and docosahexaenoic acids. The polar lipidome identified by HILIC LC-MS allowed identifying 304 different lipid species. These species belong to the phosphatidylcholine (103 lipid species), lysophosphatidylcholine (35), phosphatidylethanolamine (71), lysophosphatidylethanolamine (20), phosphatidylglycerol (13), lysophosphatidylglycerol (5), phosphatidylinositol (15), lysophosphatidylinositol (3), phosphatidylserine (6) lysophosphatidylserine (1), and sphimgomyelin (32) classes. This was the first time that the dolphin plasma phospholipid profile was characterized, providing a knowledge that will be important to further understand lipid metabolism and physiological regulation in small cetaceans. Furthermore, this study proved the practicability of the use of plasma lipid profiling for health assessment in marine mammals under human care.

Macroalgae aquaculture is thriving, and kelps such as Saccharina latissima, are now farmed over different production sites across Europe. Macroalgae production in integrated multi-trophic aquaculture setups is gaining relevance due to increased biomass yield and bioremediation potential. Nevertheless, there is a substantiated need to understand how this type of production shapes biomass composition. The present study evaluated the influence of time of harvesting and proximity to nutrient source (fish farm sea cages) on the elemental and biochemical composition of S. latissima, with special emphasis on its lipid content. Overall, the differences recorded for elemental, biochemical and lipid composition of S. latissima occurred consistently with harvesting period rather than with the distance to fish farm sea cages, evidencing that farming kelp in integrated multi-trophic aquaculture setups does not compromise nutritional quality while promoting increased biomass yield. Elemental and biochemical composition differed between kelp biomass harvested in April and those harvested in May and June, with lower C, H and carbohydrates, and higher ash contents in the later. Fatty acid profile analysis revealed an increase in monounsaturated fatty acids along the harvesting period, as well as a decrease in n-3 fatty acids with a concomitant increase of the n-6/n-3 ratio from April to May. Statistical analysis of the polar lipidome identified by liquid chromatography-mass spectrometry revealed differences in specific lipid signatures, displaying a perfect discrimination between harvesting periods. The discrimination between samples from reference and integrated multi-trophic aquaculture sites was only observed for kelp biomass harvested in June. Contributing particularly to these differences are betaine lipids, more abundant in the two later time points, and some lysolipid species, especially abundant in June. These findings will allow a more integrated look at macroalgae production under integrated multi-trophic aquaculture framework, thus promoting the systematization of farming practices that may enhance yield with biochemical quality to match the increasing demands by the food industry and sustainable biorefinary pipeline.

Brown seaweeds are known to present components with appealing bioactive properties eliciting great interest for industrial applications. However, their lipid content is generally disregarded beyond their fatty acid (FA) composition. This study thoroughly characterized the lipid profile of two brown seaweeds collected from Portuguese coast, the native Bifurcaria bifurcata and the invasive Sargassum muticum species, and bioprospecting for antioxidant activity. An integrated state-of-the-art approach including gas chromatography-mass spectrometry (GC&ndash, MS) and liquid chromatography-mass spectrometry (HILIC&ndash, ESI-MS/MS), allowed a comprehensive picture of FA and polar lipid content. Polar lipid profile of B. bifurcata and S. muticum included 143 and 217 lipid species respectively, distributed between glycolipids, phospholipids, and betaine lipids. Some of the lipid species found have been assigned biological activity and contain of n-3 and n-6 FA. Sargassum muticum presented the highest n-3 FA content. Low concentrations of extracts of both seaweeds displayed antioxidant activity, with S. muticum presenting more promising results. These findings contribute to the nutritional and industrial exploitation of both seaweeds, highlighting their relevance as viable sources of bioactive and added-value compounds. Sargassum muticum presented interesting lipid composition and bioactivity, which may represent an accessible opportunity for the exploitation of this invasive seaweed, especially taking advantage of Sargassum blooms.

The aquaculture of macroalgae for human consumption and other high-end applications is experiencing unprecedented development in European countries, with the brown algae Saccharina latissima being the flag species. However, environmental conditions in open sea culture sites are often unique, which may impact the biochemical composition of cultured macroalgae. The present study compared the elemental compositions (CHNS), fatty acid profiles, and lipidomes of S. latissima originating from three distinct locations (France, Norway, and the United Kingdom). Significant differences were found in the elemental composition, with Norwegian samples displaying twice the lipid content of the others, and significantly less protein (2.6%, while French and UK samples contained 6.3% and 9.1%, respectively). The fatty acid profiles also differed considerably, with UK samples displaying a lower content of n-3 fatty acids (21.6%), resulting in a higher n-6/n-3 ratio. Regarding the lipidomic profile, samples from France were enriched in lyso lipids, while those from Norway displayed a particular signature of phosphatidylglycerol, phosphatidylinositol, and phosphatidylcholine. Samples from the UK featured higher levels of phosphatidylethanolamine and, in general, a lower content of galactolipids. These differences highlight the influence of site-specific environmental conditions in the shaping of macroalgae biochemical phenotypes and nutritional value. It is also important to highlight that differences recorded in the lipidome of S. latissima make it possible to pinpoint specific lipid species that are likely to represent origin biomarkers. This finding is relevant for future applications in the field of geographic origin traceability and food control.

Methylated xanthines (methylxanthines) are available from a significant number of different botanical species. They are ordinarily included in daily diet, in many extremely common beverages and foods. Caffeine, theophylline and theobromine are the main methylxanthines available from natural sources. The supposedly relatively low toxicity of methylxanthines, combined with the many beneficial effects that have been attributed to these compounds through time, generated a justified attention and a very prolific ground for dedicated scientific reports. Methylxanthines have been widely used as therapeutical tools, in an intriguing range of medicinal scopes. In fact, methylxanthines have been/were medically used as Central Nervous System stimulants, bronchodilators, coronary dilators, diuretics and anti-cancer adjuvant treatments. Other than these applications, methylxanthines have also been hinted to hold other beneficial health effects, namely regarding neurodegenerative diseases, cardioprotection, diabetes and fertility. However, it seems now consensual that toxicity concerns related to methylxanthine consumption and/or therapeutic use should not be dismissed. Taking all the knowledge and expectations on the potential of methylxanthines into account, we propose a systematic look at the past and future of methylxanthine pharmacologic applications, discussing all the promise and anticipating possible constraints. Anyways, methylxanthines will still substantiate considerable meaningful research and discussion for years to come.

Breast cancer is one of the most common malignancies affecting women worldwide. However, despite its incidence trends have increased, the mortality rate has significantly decreased. The primary concern in any cancer treatment is the oncological outcome but, in the case of breast cancer, the surgery aesthetic result has become an important quality indicator for breast cancer patients. In this sense, an adequate surgical planning and prediction tool would empower the patient regarding the treatment decision process, enabling a better communication between the surgeon and the patient and a better understanding of the impact of each surgical option. To develop such tool, it is necessary to create complete 3D model of the breast, integrating both inner and outer breast data. In this review, we thoroughly explore and review the major existing works that address, directly or not, the technical challenges involved in the development of a 3D software planning tool in the field of breast conserving surgery.

T-cell division is central to maintaining a stable T-cell pool in adults. It also enables T-cell expansion in neonates, and after depletion by chemotherapy, bone marrow transplantation, or infection. The same signals required for T-cell survival in lymphoreplete settings, IL-7 and T-cell receptor (TCR) interactions with self-peptide MHC (pMHC), induce division when T-cell numbers are low. The strength of reactivity for self-pMHC has been shown to correlate with the capacity of T cells to undergo lymphopenia-induced proliferation (LIP), in that weakly self-reactive T cells are unable to divide, implying that T-cell reconstitution would significantly skew the TCR repertoire toward TCRs with greater self-reactivity and thus compromise T-cell diversity. Here, we show that while CD4+ T cells with low self-pMHC reactivity experience more intense competition, they are able to divide when present at low enough cell numbers. Thus, at physiological precursor frequencies CD4+ T cells with low self-pMHC reactivity are able to contribute to the reconstitution of the T-cell pool.

Automatic segmentation of breast is an important step in the context of providing a planning tool for breast cancer conservative treatment, being important to segment completely the breast region in an objective way; however, current methodologies need user interaction or detect breast contour partially. In this paper, we propose a methodology to detect the complete breast contour, including the pectoral muscle, using multi-modality data. Exterior contour is obtained from 3D reconstructed data acquired from low-cost RGB-D sensors, and the interior contour (pectoral muscle) is obtained from Magnetic Resonance Imaging (MRI) data. Quantitative evaluation indicates that the proposed methodology performs an acceptable detection of breast contour, which is also confirmed by visual evaluation.

Patient-specific surgical predictions of Breast Conserving Therapy, through mechano-biological simulations, could inform the shared decision making process between clinicians and patients by enabling the impact of different surgical options to be visualised. We present an overview of our processing workflow that integrates MR images and three dimensional optical surface scans into a personalised model. Utilising an interactively generated surgical plan, a multi-scale open source finite element solver is employed to simulate breast deformity based on interrelated physiological and biomechanical processes that occur post surgery. Our outcome predictions, based on the pre-surgical imaging, were validated by comparing the simulated outcome with follow-up surface scans of four patients acquired 6 to 12 months post-surgery. A mean absolute surface distance of 3.3i¾?mm between the follow-up scan and the simulation was obtained.

Rett syndrome (RS) is one of the best human models to study movement disorders. Patients evolve from a hyperkinetic to a hypokinetic state, and a large series of abnormal movements may be observed along their lives such as stereotypies, tremor, chorea, myoclonus, ataxia, dystonia, and rigidity. The aim of this work was to analyze movement disorders in RS patients with a detected MECP2 mutation, as well as their correlation with genotype, in a clinically and genetically well-characterized sample of patients, and thus contribute to redefine the clinical profile of this disease. In this study, we included 60 patients with detected MECP2 mutations. These were categorized and grouped for analysis, according to (1) type of change (missense or truncating, including nonsense and frameshift but also large deletions) and (2) location of the mutation. Differences were found concerning the frequency of independent gait, dystonia, type of tremor, and global score severity when comparing the group of patients with missense and truncating mutations. We also found differences in the presence, distribution, severity, or type of movement disorders in the two groups of patients according to the median duration of the disease (less than 60 months; 60 months or more). We conclude that movement disorders seem to reflect the severity and rate of progression of Rett disorder, patients with truncating mutations presenting a higher rate and more severe dystonia and rigid-akinetic syndrome, when comparing groups with similar time of disease evolution.

How many times have you been to the doctor and was told that a balanced lifestyle is key for a healthy, disease-free, long life? How many times did genetics take the blame? But do we really know whether genetic risk associated with complex diseases can be offset by behavioral changes? A recent study at the New England Journal of Medicine now brings us some answers.Image from iStock.com/udraView Large Image | View Hi-Res Image | Download PowerPoint SlideThe idea that most human diseases are the result of a complex interplay between genes and environment is not new. Complex genetic disorders, those in which the effects of multiple genes are at play and clear-cut patterns of inheritance are difficult to pinpoint, are particularly influenced by lifestyle and environmental factors. Take heart disease as an example. While one’s own risk of cardiovascular disease increases by 3- to 5-fold if a person’s parent is diseased, factors such as diet, physical activity, tobacco use, environmental stress, and host factors such as age and sex also play a significant role in the risk of developing disease. The problem is that quantifying the contribution of each of these factors is tricky. Khera et al. (2016)xKhera, A.V., Emdin, C.A., Drake, I., Natarajan, P., Bick, A.G., Cook, N.R., Chasman, D.I., Baber, U., Mehran, R., Rader, D.J. et al. N. Engl. J. Med. 2016; CrossrefSee all References)Khera et al. (2016) set up to try to answer the billion dollar question: can a healthy lifestyle compensate for a strong genetic risk of developing disease?Genetic risk in complex diseases results from the combination of incremental effects of a large number of DNA sequence polymorphisms. In the case of coronary artery disease and myocardial infarct—a common and severe complication of cardiac disease—over 50 independent loci have been associated with risk of disease in genome-wide analyses. The aggregate of these risk genes provides has proved useful in defining genetic susceptibility to cardiovascular events. Khera et al. make use of this strategy to stratify the ∼60,000 subjects in their prospective study into three genetic risk categories: low, intermediate, and high. Then they ask whether baseline heathy lifestyle choices impact the risk of developing coronary events and atherosclerosis. The definition of healthy lifestyle was drawn from the strategic goals of the American Heart Association (AHA) to reduce the risk of cardiovascular in the general population and include no current smoking, no obesity, physical activity at least once weekly, and a healthy diet pattern. Importantly, the subjects in the study were previously enrolled in independent studies and represent three completely independent cohorts, what helps to power their analyses.As one would expect, subjects with high polygenic risk score were at much higher risk of developing coronary events than those at low genetic risk. What may come as a surprise is that further dividing subjects of different genetic risk groups according to their lifestyle choices shows that those who make healthy lifestyle choices present ∼45%–47% lower relative risk of developing coronary events. Even subjects at high genetic risk have their risk reduced when they make healthy lifestyle choices. The association also seems to hold true in men and women, and it does not seem to be under strong influence of ancestry. Moreover, not only the risk of adverse coronary events is reduced by adherence to healthy lifestyle but also coronary artery calcification, which is a prelude to more severe disease.Does that mean that going to the gym and eating fresh fruit, vegetables, grains, nuts and fish (while avoiding saturated fat, red meat and sugar-sweetened beverages) can counteract completely the effect of genetic inheritance? Certainly not. The new study clearly shows that high genetic risk is a strong and independent risk factor that increases the risk of coronary events and atherosclerosis. What perhaps is unexpected is that genetics and healthy lifestyle choices seem to be independently contributing to the susceptibility of heart disease. While it is true that people with high genetic risk benefit the most from healthy choices, the positive effect is also seen in those with intermediate and low genetic risk. In other words, healthy lifestyle benefits everyone, and high genetic risk is not necessarily deterministic of disease outcome.What do these findings mean from a basic biology perspective? Will we be able to get passed associations and begin to understand relationships of causality between healthy habits, genetics and disease? Will we be able to move beyond generic prescriptions of diet and exercise and tailor one’s choices to his or her genetics? Recent data have already showed that the response to food, for instance, is highly individualized when it comes to metabolism and glucose response (Zeevi et al., 2015xPersonalized Nutrition by Prediction of Glycemic Responses. Zeevi, D., Korem, T., Zmora, N., Israeli, D., Rothschild, D., Weinberger, A., Ben-Yacov, O., Lador, D., Avnit-Sagi, T., Lotan-Pompan, M. et al. Cell. 2015; 163: 1079–1094Abstract | Full Text | Full Text PDF | PubMed | Scopus (69)See all ReferencesZeevi et al., 2015), speaking to the need a personalized approaches for nutrition and medicine. Further, can the risk to other complex diseases be modified by behavioral changes? While we eagerly wait for answers to some of these questions, perhaps it would be wise to stick with carrots.

Diet-induced changes in the lipid composition of mitochondrial membranes have been shown to influence physiological processes. However, the modulation effect of diet on mitochondrially-active drugs has not yet received the deserved attention. Our hypothesis is that modulation of membrane dynamics by diet impacts drug-effects on liver mitochondrial functioning. In a previous work, we have shown that a diet rich in rapeseed oil altered mitochondrial membrane composition and bioenergetics in Wistar rats. In the present work, we investigated the influence of the modified diet on hepatic mitochondrial activity of two drugs, menadione and nimesulide, and FCCP, a classic protonophore, was used for comparison. The results showed that the effects of menadione and nimesulide were less severe on liver mitochondria for rats fed the modified diet than on rats fed the control diet. A specific effect on complex I seemed to be involved in drug-induced mitochondria dysfunction. Liver mitochondria from the modified diet group were more susceptible to nimesulide effects on MPT induction. The present work demonstrates that diet manipulation aimed at modifying mitochondrial membrane properties alters the toxicity of mitochondria active agents. This work highlights that diet may potentiate mitochondrial pharmacologic effects or increase drug-induced liabilities. The project was supported by the Foundation for Science and Technology (FCT, Portugal) with FEDER/COMPETE/National Budget funds (research grants PTDC/QUI-QUI/101409/2008 to P.J.O., PTDC/QUI-BIQ/103001/2008 to A.S.J. and PEst-C/SAU/LA0001/ 2013-2014 to the CNC). J.P.M. and A.M.S acknowledge FCT for Ph.D. grants SFRH/BD/37626/2007 and PTDC/AGR-ALI/108326/ 2008 respectively.

Diet is directly related with physiological alterations occurring at a cell and subcellular level. However, the role of diet manipulation on mitochondrial physiology is still largely unexplored. Aiming at correlating diet with alterations of mitochondrial membrane composition and bioenergetics, Wistar-Han male rats were fed for 11, 22 and 33 days with a rapeseed oil-based diet and mitochondrial bioenergetics, and membrane composition were compared at each time point with a standard diet group. Considerable differences were noticed in mitochondrial membrane lipid composition, namely in terms of fatty acyl chains and relative proportions of phospholipid classes, the modified diet inducing a decrease in the saturated to unsaturated molar ratio and an increase in the phosphatidylcholine to phosphatidylethanolamine molar ratio. Mass spectrometry lipid analysis showed significant differences in the major species of cardiolipin, with an apparent increased incorporation of oleic acid as a result of exposure to the modified diet. Rats fed the modified diet during 22 days showed decreased hepatic mitochondrial state 3 respiration and were more susceptible to Ca2+-induced transition pore opening. Rapeseed oil-enriched diet also appeared to promote a decrease in hydroperoxide production by the respiratory chain, although a simultaneous decrease in vitamin E content was detected. In conclusion, our data indicate that the rapeseed oil diet causes negative alterations on hepatic mitochondrial bioenergetics, which may result from membrane remodeling. Such alterations may have an impact not only on energy supply to the cell, but also on drug-induced hepatic mitochondrial liabilities. The project was supported by the Foundation for Science and Technology with FEDER/COMPETE/National Budget funds (research grants PTDC/QUI–QUI/101409/2008 to P. J. O., PTDC/QUI-BIQ/103001/2008 to A. S. J. and strategic grant PEst- C/SAU/LA0001/2011to the CNC). J. P. M. and A. M. S. acknowledge FCT for Ph.D. grants SFRH/BD/37626/2007 and PTDC/AGRALI/ 108326/2008, respectively.

Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. 31P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MEN's ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity.

Increasing global demand for food has generated a need for new strategies to feed a fast-growing population. Oceans appear as a sustainable solution, providing alternative sources of food such as macroalgae. These sea vegetables have high nutritional value and provide functional and health benefits. The sugar kelp Saccharina latissima is an emerging edible seaweed used mainly for human consumption. Although much is known about its biochemical compositions its lipidome remains unexplored. The aim of the present study was to characterize the polar lipid profile of S. latissima using a lipidomic-mass spectrometry HILIC-LC-MS based analysis. This approach allowed the identification of 197 molecular species of polar lipids, including glycolipids, phospholipids and betaine lipids. Several molecular species identified are carriers of polyunsaturated fatty acids with nutritional value and have been reported with anti-inflammatory, anti-microbial and anti-proliferative activity. Saccharina latissima is an emerging candidate to promote blue biotechnology inspired by the ocean.