Decarbonizing the energy supply one pandemic at a time.

This study explores different energy consumption vectors during the first year of the COVID-19 pandemic in Portugal. Most of the workforce started working from home and resource consumption significantly shifted towards the domestic sector. The ensuing confinement protocols caused a shift in everyday life, which in turn significantly altered the energy supply and demand landscape. This event, although catastrophic in terms of loss of human life and economic development, can provide us with valuable data to study the potential of new strategies to achieve EU 2050 Energy goals. It was investigated whether the pandemic has opened a path and provided us with a partial answer to decarbonization in the form of home office practices as a possible energy efficiency measure. The present study shows that, in Portugal, there was a 15.7% reduction of primary energy consumption (accounting for electricity, natural gas and transport fuels) compared to 2019. The data suggest that actions targeting reduced mobility, such as home office practices and the decentralization of the workforce, could be a relevant energy efficiency measure.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Influence of age and physical exercise on sirtuin activity in humans.

Sirtuins are NAD+-dependent lysine deacetylases. Sirtuins acquired worldwide attention because of their ability to increase yeast, flies, worms and mice lifespan. Recently, this assumption has been challenged. However, their beneficial role on the quality of ageing is widely accepted. In this work we aimed to study how and if sirtuins expression and activity levels varies in function of age and, in the case of young subjects, of exercise. Fifteen blood donors of different ages and fifteen athletes of the Italian rowing male team were enrolled and peripheral blood mononuclear cells (PBMCs) isolated from blood samples. Our results show that sirtuins deacetylases activity measured in PBMCs increases from 18 to 40 years of age and then decreases during the following 20 years. Moreover, physical exercise in professional athletes can upregulate sirtuin activity. Thus, for the first time in humans, we demonstrate that sirtuin activity is a function of age and can be altered through physical exercise.

An integrated analysis of COVID-19 impacts on energy and environment: Lessons learnt.

Carbon neutrality, sustainable development and reducing our impact on the environment is the top priority in future measures. The COVID-19 pandemic brought challenges to every sector at a global scale but can provide valuable insight to reach these goals. The main objective of this work is to provide an integrated analysis of the impact of the COVID-19 pandemic, focused on energy and its related aspects, i.e., environment and costs. Mainland Portugal was used as a case study and two years were analysed, one pre pandemic (2019) and another post pandemic (2020). In 2020, the majority of sectors - Transport, Services, Industry and Agriculture & Fisheries - show a reduction of energy consumption, atmospheric emissions, carbon footprint and related monetary and social costs. In contrast, the Domestic sector presents an overall increase, with maximums of 25.4% in electricity consumption (during Spring), 0.72% in the PM10 (particulate matter) and NOx (nitrogen dioxides) emissions (in Summer), and 2.9% in carbon footprint (in Spring). The integrated analysis proposed in this work was crucial to identify the paths to a post pandemic world focused on the different aspects of sustainability - new concepts of mobility and workplace, as well as increased investment in energy performance and renewable energy sources. This study showed that changing our energy consumption patterns could significantly affect future greenhouse gas emissions, and contribute to the sustainable growth of the economy, while maintaining good progress towards climate-neutral goals.

Lipido-sterolic extract of Serenoa repens (LSESr, Permixon) treatment affects human prostate cancer cell membrane organization.

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.

Patient preference for sustained-release versus standard paracetamol (acetaminophen): a multicentre, randomized, open-label, two-way crossover study in subjects with knee osteoarthritis.

Guidelines for osteoarthritis (OA) management recommend paracetamol (acetaminophen) as the most appropriate first-line analgesic for mild to moderate pain. Standard paracetamol requires four times daily dosing. Drug compliance and convenience are inversely related to daily dose frequency. Compliance is a pivotal component of the successful management of OA pain and is influenced by patient preferences or beliefs. The added convenience of three times daily dosing may enhance compliance and, therefore, pain relief. This multicentre, randomized, open-label, two-way crossover, phase IV study is the first to evaluate patient preference with a sustained-release paracetamol tablet formulation designed for three times daily dosing. Compared with standard paracetamol tablets dosed four times daily, the sustained-release formulation was preferred in a 2:1 ratio, provided better overall joint pain relief, resulted in higher levels of satisfaction in subjects with OA of the knee and has the potential to improve patient compliance and, therefore, pain control.

Increased apoptosis, Huntingtin inclusions and altered differentiation in muscle cell cultures from Huntington's disease subjects.

Mutated huntingtin (htt) is ubiquitously expressed in tissues of Huntington's disease (HD) patients. In the brain, the mutated protein leads to neuronal cell dysfunction and death, associated with formation of htt-positive inclusions. Given increasing evidence of abnormalities in HD skeletal muscle, we extensively analyzed primary muscle cell cultures from seven HD subjects (including two unaffected mutation carriers). Myoblasts from presymptomatic and symptomatic HD subjects showed cellular abnormalities in vitro, namely mitochondrial depolarization, cytochrome c release, increased caspase-3, -8, and -9 activities, and defective cell differentiation. Another notable feature was the formation of htt inclusions in differentiated myotubes. This study helps to advance current knowledge about the downstream effects of the htt mutation in human tissues. Further applications may include drug screening using this human cellular model.

The metabolism-dependent maintenance of cell volume and ultrastructure in slices of Morris hepatoma 3924A.

The changes in water and electrolyte content of slices of Morris hepatoma 3924A induced by various conditions of incubation have been compared with the ultrastructural appearance of the tissue. Incubation at 1 degrees led to an increase of water, Na+, and Cl- content and to a loss of K+. There was simultaneous increase in size of the cells and intercellular spaces, loss of junctional complexes, increase in the number of microvilli, and fragmentation and dilation of the cytocavitary network. Subsequent incubation at 38 degrees in oxygenated medium led to a substantial reversal of all of these changes of composition and structure, which was well advanced within 10 min and largely complete by 60 min. The presence of 20 mM glucose in the medium somewhat enhanced the degree of recovery. A reduction of cell volume and intercellular spaces was evident both from the electron microscopic observations and measurements of the volume of inulin distribution. The presence of ouabain inhibited the net accumulation of K+ and much of the Na+ extrusion, but permitted about 50% of the net extrusion of water (accompanied by Na+ and Cl-) and had little effect on the ultrastructural recovery. The presence of glucose increased the resistance of volume and structural recovery of ouabain without releasing the inhibition of K+ accumulation. A marked feature of the recovering tissues was the Golgi apparatus, which assumed an appearance suggestive of increased activity when water extrusion was active. In slices using only endogenous substrate, cyanide and (to a lesser extent) oligomycin greatly inhibited the recovery of volume and structure. The presence of glucose permitted some recovery in the presence of cyanide. The control of cell volume in hepatoma 3924A appears to involve two separate components of water transport, one of which is sensitive, and one insensitive to ouabain. The ouabain-insensitive component appears to be especially related to the recovery of cell ultrastructure after incubation at 1 degrees, to be more sensitive to paucity of adenosine 5'-triphosphate, and to proceed by secretion of water, Na+, and Cl- into vesicles that fuse with the Golgi apparatus. This mechanism may be related to that for bile secretion in normal liver. The ouabain-sensitive component of water transport is a function of the mechanism for the coupled transport of Na+ and K+.

Interaction of Na+ and K+ transport with aerobic energy metabolism in slices of Morris hepatoma 3924A.

Addition of increasing concentrations of glucose to slices of Morris hepatoma 3924A greatly stimulated aerobic lactate production and reduced respiration by 20%. Neither the adenine nucleotide content of the slices nor the calculated rate of adenosine 5'-triphosphate synthesis was altered. Ouabain reduced the rate of O2 uptake (by 20 to 25%) and of aerobic lactate production (by 25 to 50%) without affecting adenine nucleotide contents. The reduction by ouabain of the calculated rate of adenosine 5'-triphosphate synthesis was similar whether the slices were utilizing only endogenous substrate or exogenous glucose also. Raising the medium K+ concentration (and correspondingly reducing Na+) partially overcame the inhibition of ion transport by ouabain and partially restored the rates of respiration and aerobic lactate production toward control levels. Electron microscopic observations of mitochondria within the slices incubated under different conditions showed variations in configuration between "orthodox," "condensed" and degenerating forms. Slices preincubated at 1 degrees showed mitochondria in the condensed form: they were restored to the orthodox configuration during incubation at 38 degrees in oxygenated medium. Oligomycin and glucose enhanced the transition, but ouabain reduced the number of mitochondria undergoing the change. The results suggest that in hepatoma 3924A utilization of adenosine 5'-triphosphate by ion transport exerts a simultaneous control of both respiration and aerobic glycolysis, which is presumably mediated by alterations in the availability of adenosine 5-diphosphate. The mitochondria undergo conformational transitions under conditions likely to affect local availability of adenosine 5'-diphosphate within cell compartments, but the transitions are not all externally added adenosine diphosphate on isolated mitochondria.

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase.

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Ouabain-induced cytoplasmic vesicles and their role in cell volume maintenance.

Cellular swelling is controlled by an active mechanism of cell volume regulation driven by a Na(+)/K(+)-dependent ATPase and by aquaporins which translocate water along the osmotic gradient. Na(+)/K(+)-pump may be blocked by ouabain, a digitalic derivative, by inhibition of ATP, or by drastic ion alterations of extracellular fluid. However, it has been observed that some tissues are still able to control their volume despite the presence of ouabain, suggesting the existence of other mechanisms of cell volume control. In 1977, by correlating electron microscopy observation with ion and water composition of liver slices incubated in different metabolic conditions in the presence or absence of ouabain, we observed that hepatocytes were able to control their volume extruding water and recovering ion composition in the presence of ouabain. In particular, hepatocytes were able to sequester ions and water in intracellular vesicles and then secrete them at the bile canaliculus pole. We named this "vesicular mechanism of cell volume control." Afterward, this mechanism has been confirmed by us and other laboratories in several mammalian tissues. This review summarizes evidences regarding this mechanism, problems that are still pending, and questions that need to be answered. Finally, we shortly review the importance of cell volume control in some human pathological conditions.

Muscle LIM protein/CSRP3: a mechanosensor with a role in autophagy.

Muscle LIM protein (MLP) is a microtubule-associated protein expressed in cardiac and muscle tissues that belongs to the cysteine-rich protein (CSRP/CRP) family. MLP has a central role during muscle development and for architectural maintenance of muscle cells. However, muscle cells rely on autophagy during differentiation and for structural maintenance. To study the role of MLP in autophagy, we have used C2C12 mouse myoblasts silenced or overexpressing MLP. Our results show that MLP contributes to the correct autophagosome formation and flux by interacting with LC3 as demonstrated by co-immunoprecipitation and PLA assay. In fact, MLP silencing results in decreased LC3-II staining and absent degradation of long-lived proteins. Moreover, MLP silencing impaired myoblasts differentiation as measured by decreased expression of MyoD1, MyoG1 and myosin heavy chain. Ultrastructural analysis revealed the presence of large empty autophagosomes in myoblasts and multimembranous structures in myotubes from MLP-silenced clones. Impaired autophagy in MLP-silenced cells resulted in increased susceptibility to apoptotic cell death. In fact, treatment of MLP-silenced C2C12 myoblasts and myotubes with staurosporine resulted in increased caspase-3 and PARP cleavage as well as increased percentage of cell death. In conclusion, we propose that MLP regulates autophagy during muscle cell differentiation or maintenance through a mechanism involving MLP/LC3-II interaction and correct autophagosome formation.

Effects of ouabain and chloride-free medium on isoosmotic volume control and ultrastructure of hepatocytes in primary culture.

Rat hepatocytes in primary monolayer culture have been studied by a combination of physiological and morphological approaches under conditions affecting ion transport and cell volume. A concentration of ouabain completely inhibiting the coupled transport of Na+ and K+ had little effect on cell volume, as indicated by cell water content, but induced the formation of many vesicles in the cytoplasm. Apparent fusion of vesicles was often observed. By itself, replacement of medium Cl by NO3- had little effect on cell volume or morphology. However, when NO3- replaced Cl- in the presence of ouabain the cells swelled and the numbers and size of vesicles were much reduced. The vesicles accumulating in the presence of ouabain showed a yellow fluorescence after the cells were loaded with acridine orange, implying that the vesicular contents were acidic. Total fluid-phase endocytosis, determined by uptake of Lucifer yellow, was not affected by ouabain or the absence of Cl-. However, ouabain considerably retarded the subsequent release of Lucifer yellow; this suggests that the dye originally taken into endocytotic vesicles became diluted by mixing with contents of ouabain-induced vesicles, an explanation consistent with the vesicle fusion seen by electron microscopy. The Cl-free medium also retarded Lucifer yellow efflux, to the same extent as ouabain, and the effects of the two treatments were not additive. These observations are consistent with the activity in hepatocytes of an ouabain-resistant, Cl(-)-dependent mechanism for cell volume control. It is suggested that this depends on the accumulation of water into acidic vesicles, which is driven by the Cl(-)-coupled activity of the vacuolar ATPases of the organelles, followed by exocytotic expulsion of their contents.

Identification of an estrogen-mediated deoxyribonucleic acid-binding independent transactivation pathway on the epidermal growth factor receptor gene promoter.

To investigate the estrogenic effects on the transcriptional regulation of the epidermal growth factor (EGF) receptor (EGFR) gene, we assayed its promoter ability to direct transcription of the luciferase reporter gene after transfection into HeLa cells. Our studies demonstrated a dose-dependent activation of the EGFR gene transcription by ligand-bound estrogen receptor alpha (ERalpha). This action was retained by the 36-bp core promoter fragment and did not require the receptor DNA binding domain, as demonstrated by analyzing the role of ERalpha deletion mutants on EGFR gene promoter-derived constructs. The 36-bp promoter fragment does not contain an estrogen response element but an imperfect thyroid hormone response element half-site that overlaps the Sp1 binding site. ERalpha does not bind this imperfect thyroid hormone response element half-site but is able to enhance binding of Sp1 to its site, in gel mobility shift assays, suggesting that the mechanism by which the receptor stimulated the transcription involved protein-protein interactions that replaced DNA binding. To explain this action, we propose a model in which induction of the EGFR gene expression by estrogens in HeLa cells is dependent upon the formation of a transcriptionally active ERalpha-Sp1 complex that binds to the GC-rich (Sp1) region of the minimal promoter.

Catecholamine-induced cardiomyopathy in multiple endocrine neoplasia. A histologic, ultrastructural, and biochemical study.

A catecholamine-induced dilated cardiomyopathy is reported in a patient with multiple endocrine neoplasia, type 3. A histologic and ultrastructural study has been undertaken in cardiac biopsy samples, together with determination of myocardial Ca++ and cellular membrane fatty acids. Contraction band necrosis of cardiocytes with supercontraction of sarcomeres progressing to myofibrolysis and increased levels of myocardial Ca++ have been found as morphologic and biochemical abnormalities, respectively. No lipoperoxidation of cellular membranes or an alpha-adrenergic mediated reduction of coronary supply could be recognized in the study. We indicate a receptor-mediated intracellular Ca++ overload as the main abnormality responsible for myocardial impairment.

Hormonal regulation of cytokine release by human fetal membranes at term gestation: effects of oxytocin, hydrocortisone and progesterone on tumour necrosis factor-alpha and transforming growth factor-beta 1 output.

Inflammatory cytokines can play an important role in the biomolecular processes leading to labour by regulating prostaglandin production in intrauterine tissues. In the setting of intrauterine infection, an increased production of these cytokines by placenta, decidua and fetal membranes occurs and is responsible for the onset and maintenance of preterm labour. However, the factors involved in the control of cytokine release by these tissues in normal pregnancy at term are still largely unknown. We investigated the possibility that the synthesis and release of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) by human fetal membranes at term gestation is regulated by several hormones potentially involved either in the maintenance of pregnancy or in the parturitional process. In the present study, the effects of hydrocortisone, progesterone and oxytocin on TNF-alpha and TGF-beta1 release by explants of fetal membranes at term gestation were evaluated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the effect of the above hormones on mRNA expression; TNF-alpha and TGF-beta1 release in culture medium was quantitifed by ELISA assays. Results show that both tissue mRNA expression for TNF-alpha and TNF-alpha release in culture medium were significantly increased by oxytocin, but not by hydrocortisone and progesterone. On the contrary, all the hormones tested increased both tissue TGF-beta1 mRNA expression and release in culture medium. These findings suggest that TNF-alpha and TGF-beta1 production by human fetal membranes in uncomplicated pregnancy at term is selectively modulated by oxytocin, hydrocortisone and progesterone.

Reversible dilated cardiomyopathy due to growth hormone deficiency.

An unusual case of severe dilated cardiomyopathy is described in a woman with postpartum hypopituitary function, which was characterized by marked decrease of cardiac myofibrils (decreased ratio of myofibrillar volume:cell volume) and somatomedin C deficiency. The patient was treated with a daily intramuscular dose of 4 UI of recombinant human growth hormone for 3 months. Clinical and laboratory assessment and follow-up consisted of noninvasive (echocardiograms, Doppler study, and clinical laboratory tests) and invasive procedures (angiography and endomyocardial biopsy). Morphologic study included optical and electron microscopic examinations and morphometric analysis. The patient exhibited a relevant improvement of cardiac function after recombinant human growth hormone administration. The clinical improvement was confirmed by normalization of electrocardiographic voltages, increased myocardial mass, angiography, and optical and electron microscopic examination, which showed a dramatic recovery of myofibrillar content in myocardiocytes. Furthermore, withdrawal of growth hormone was followed by reduction of electrocardiographic voltages and myocardial mass as well as by impairment of left ventricular function. The authors suggest that there are forms of dilated cardiomyopathy that may benefit dramatically by growth hormone administration. These forms should be identified by growth hormone/somatomedin C axis deficiency and by decrease in myofibrillar content at endomyocardial biopsy.

Immunochemical and ultrastructural study of multiple myeloma with a heavy chain protein in the serum.

A patient with multiple myeloma had antigenically related monoclonal Fc-gamma fragments and complete IgG-kappa molecules in the serum. The urine contained only Fc-gamma fragments in the absence of Bence-Jones protein. The two distinct M-components in the serum showed electrophoretic identity but could be separated by chromatography. The simultaneous presence of complete monoclonal IgG molecules and Fc-gamma fragments, though difficult to detect, could be a frequent occurrence in multiple myeloma, and it could be defined as 'double paraproteinaemia'. A detailed ultrastructural study was performed in this case and showed fibril bundles being released from the malignant plasma cells; such fibrils could be the supramolecular organisation of the neosynthesised heavy chain fragments.

Managed care in the Northern California Kaiser Permanente health care system.

Kaiser Permanente in Northern California is a group model health maintenance organization (HMO) that contracts with The Permanente Medical Group, Inc. (TPMG) to provide care for health plan members. TPMG is the largest (for-profit) private group practice in the world, with 3600 physicians in Northern California, including 62 urologists, providing care to 2.5 million health plan members. Recent changes in health care financing and delivery mandate an HMO-physician relationship which addresses issues of cost, quality of care, and the needs of physicians.