Reconnecting the Broken Bonds: Environment, Politics, Economics and the State of the World

An ecosystem theoretical and practical framework is posited for the evaluation and planning of advocacy, communication, public policies, research and teaching programmers, intertwining four dimensions of being-in-the-world (intimate, interactive, social and biophysical), as they combine, as donors and recipients, to induce the events (deficits/assets), cope with consequences (desired/undesired) and contribute for change (potential outputs). Earth’s regeneration and mankind’s regeneration, as faces of the same coin, are addressed simultaneously, in space and time, for their mutual support. Goals and new paths to reach them contemplate a set of values, norms and policies that prioritizes socio-ecological objectives, human well-being, natural and built environments, the aesthetic, ethical and cultural meaning of the existence

Living better in a better world: Guidance and counselling in an ecosystemic model of culture

Diagnosis and prognosis of current problems take into account the connections (assets) and ruptures (deficits) between the different dimensions of being-in-the-world, mutually entangled as donors and recipients: intimate; interactive; social and biophysical. Guidance and counselling consider the complex and dynamic configurations formed by the intertwining of the different dimensions, as they combine to produce the events. Cultural and epistemic backgrounds, subject-object relationships, assumptions and conflicts, are examined by heuristic-hermeneutic processes, as new support structures emerge in the socio-cultural learning niches. Problems related to education, culture, ethics, physical, social and mental well-being, natural and man-made environment are treated as ecosystemic configurations, not as separate objects of separate programmes. Values, goals, and principles are considered in the transition from a non-ecosystemic to an ecosystemic model of culture. The proposal presents not only a descriptive position, but also a normative position, a framework for the development and evaluation of public policies and research and teaching programmes, critically inquiring into the prevailing assumptions of growth, power, wealth, work and freedom

Challenges in Chagas Disease Drug Development.

The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health problem throughout Latin America. Current therapeutic options are characterised by limited efficacy, long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless, several factors have come together to create a more optimistic scenario. Drug-based research has become more systematic, with increased collaborations between the academic and commercial sectors, often within the framework of not-for-profit consortia. High-throughput screening of compound libraries is being widely applied, and new technical advances are helping to streamline the drug development pipeline. In addition, drug repurposing and optimisation of current treatment regimens, informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide an overview of the current status of Chagas disease drug development, highlight those areas where progress can be expected, and describe how fundamental research is helping to underpin the process

A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery

Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized 35 S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be obtained. We also describe strategies to fortify the system with recombinant proteins to increase import yields and block specific parts of the machinery at a number of steps. The system recapitulates all the steps of the pathway, including mono-ubiquitination of PEX5L/S at the peroxisome membrane and its ATP-dependent export back into the cytosol by PEX1/PEX6. An in vitro import(/export) experiment can be completed in 24 h.We thank M. Fransen, Katholieke Universiteit-Leuven, for critical comments on the manuscript and for the plasmid encoding histidine-tagged PEX19. We thank P. van Veldhoven, Katholieke Universiteit-Leuven, and P. Maciel, Universidade do Minho, for the expression plasmids encoding prePHYH and GST-Ub, respectively. This work was funded by FEDER—Fundo Europeu de Desenvolvimento Regional through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, Portugal’s FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the projects ‘The molecular mechanism of protein import into peroxisomes’ (FCOMP-01-0124-FEDER-019731-PTDC/BIA-BCM/118577/2010), ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and ‘The molecular mechanisms of peroxisome biogenesis’ (PTDC /BEX-BC M/2311/2014) and Norte 2020—Programa Operacional Regional do Norte, under the application of the ‘Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008)’, awarded to J.E.A. T.A.R., T.F., A.F.D. and C.P.G. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

Protein transport into peroxisomes: Knowns and unknowns

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the “plunger” - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the “barrel” - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the “barrel” is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.We would like to thank Dr. Marc Fransen (KU Leuven) for his critical reading of the manuscript. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional, funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministerio da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “Themolecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020–Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008).” T.F., T.A.R., A.F.D., A.B.B., and D.B. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

The peroxisomal matrix protein translocon is a large cavity-forming protein assembly into which PEX5 protein enters to release its cargo

A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5– cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1–197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1–125)) still interacted correctly with the DTM; however, this species was largely accessible to exoge-nously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1–125)–DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138 – 639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5–DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo.This work was supported in part by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operational Pro-gramme for Competitiveness and Internationalization (POCI), Portugal 2020 and by Portuguese funds through Fundação para a Ciência e a Tec-nologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (Grant POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (Grant PTDC/BEX-BCM/2311/2014) and through Norte 2020-Programa Operacional Regional do Norte under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (Grant NORTE-01-0145-FEDER-000008). The authors declare that they have no conflicts of interest with the contents of this article

Time course and mechanisms of left ventricular systolic and diastolic dysfunction in monocrotaline-induced pulmonary hypertension

Although pulmonary hypertension (PH) selectively overloads the right ventricle (RV), neuroendocrine activation and intrinsic myocardial dysfunction have been described in the left ventricle (LV). In order to establish the timing of LV dysfunction development in PH and to clarify underlying molecular changes, Wistar rats were studied 4 and 6 weeks after subcutaneous injection of monocrotaline (MCT) 60 mg/kg (MCT-4, n = 11; MCT-6, n = 11) or vehicle (Ctrl-4, n = 11; Ctrl-6, n = 11). Acute single beat stepwise increases of systolic pressure were performed from baseline to isovolumetric (LVPiso). This hemodynamic stress was used to detect early changes in LV performance. Neurohumoral activation was evaluated by measuring angiotensin-converting enzyme (ACE) and endothelin-1 (ET-1) LV mRNA levels. Cardiomyocyte apoptosis was evaluated by TUNEL assay. Extracellular matrix composition was evaluated by tenascin-C mRNA levels and interstitial collagen content. Myosin heavy chain (MHC) composition of the LV was studied by protein quantification. MCT treatment increased RV pressures and RV/LV weight ratio, without changing LV end-diastolic pressures or dimensions. Baseline LV dysfunction were present only in MCT-6 rats. Afterload elevations prolonged tau and upward-shifted end-diastolic pressure dimension relations in MCT-4 and even more in MCT-6. MHC-isoform switch, ACE upregulation and cardiomyocyte apoptosis were present in both MCT groups. Rats with severe PH develop LV dysfunction associated with ET-1 and tenascin-C overexpression. Diastolic dysfunction, however, could be elicited at earlier stages in response to hemodynamic stress, when only LV molecular changes, such as MHC isoform switch, ACE upregulation, and myocardial apoptosis were present.Supported by Portuguese grants from FCT (POCI/SAU-FCF/60803/2004 and POCI/SAU-MMO/61547/2004) through Cardiovascular R&D Unit (FCT No. 51/94)