Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Symposium review: Fueling appetite: Nutrient metabolism and the control of feed intake.

Conceptual models developed over the past century describe 2 key constraints to feed intake (FI) of healthy animals: gut capacity and metabolic demand. Evidence that greater energy demands (e.g., greater milk production) drive a corresponding increase in caloric intake led to the dominant concept that animals "eat to energy requirements." Although this model provides reasonable initial estimates of FI, it lacks a proposed physiological basis for the control system, does not consider nutrient constraints beyond energy, and fails to explain differential energy intake responses to different fuels. To address these gaps, research has focused on mechanisms for sensing nutrient availability and providing feedback to hypothalamic centers that integrate signals to control feeding behavior. The elimination of FI response to certain nutrients by vagotomy suggests that peripheral tissues play a role in nutrient sensing. These findings and the central role of the liver in metabolic flux led to the development of the hepatic oxidation theory (HOT). According to the HOT, liver energy charge is the regulated variable that induces dietary intake changes and consequently affects whole-body energy balance. Evidence in support of HOT includes associations between hepatic energy charge and meal patterns, increased FI in response to phosphate trapping, and reduced FI in response to phosphate loading. In accordance with the HOT, infusion studies in dairy cattle have consistently demonstrated that providing fuels that either oxidize or stimulate oxidation in the liver decreases FI and energy intake to a greater extent than fuels that bypass the liver. Importantly, this holds true for glucose, which is readily oxidized by nerve cells, but is rarely taken up by the bovine liver. Although the brain integrates multiple signals including those related to gastric distention and illness, the HOT provides a physiological framework for understanding the dominant role the liver likely plays in sensing short-term energy status. Understanding this model provides insights into how to use or bypass the regulatory system to manage FI of animals.

SlS5H silencing reveals specific pathogen-triggered salicylic acid metabolism in tomato.

BACKGROUND: Salicylic acid (SA) is a major plant hormone that mediates the defence pathway against pathogens. SA accumulates in highly variable amounts depending on the plant-pathogen system, and several enzyme activities participate in the restoration of its levels. Gentisic acid (GA) is the product of the 5-hydroxylation of SA, which is catalysed by S5H, an enzyme activity regarded as a major player in SA homeostasis. GA accumulates at high levels in tomato plants infected by Citrus Exocortis Viroid (CEVd), and to a lesser extend upon Pseudomonas syringae DC3000 pv. tomato (Pst) infection. RESULTS: We have studied the induction of tomato SlS5H gene by different pathogens, and its expression correlates with the accumulation of GA. Transient over-expression of SlS5H in Nicotiana benthamiana confirmed that SA is processed by SlS5H in vivo. SlS5H-silenced tomato plants were generated, displaying a smaller size and early senescence, together with hypersusceptibility to the necrotrophic fungus Botrytis cinerea. In contrast, these transgenic lines exhibited an increased defence response and resistance to both CEVd and Pst infections. Alternative SA processing appears to occur for each specific pathogenic interaction to cope with SA levels. In SlS5H-silenced plants infected with CEVd, glycosylated SA was the most discriminant metabolite found. Instead, in Pst-infected transgenic plants, SA appeared to be rerouted to other phenolics such as feruloyldopamine, feruloylquinic acid, feruloylgalactarate and 2-hydroxyglutarate. CONCLUSION: Using SlS5H-silenced plants as a tool to unbalance SA levels, we have studied the re-routing of SA upon CEVd and Pst infections and found that, despite the common origin and role for SA in plant pathogenesis, there appear to be different pathogen-specific, alternate homeostasis pathways.

Assessing Remotely Piloted Aerial Systems in the characterization of rocky shores for oil spills environmental sensitivity mapping, northern São Paulo littoral, Brazil.

The Environmental Sensitivity Index (ESI) for oil spills was developed to assist coordinators to evaluate oil spill impact along shorelines and also to coordinate the allocation of resources during and after the incident, aiming to reduce environmental damage and consequences. Recently, Remotely Piloted Aerial Systems (RPAS) are being used in a wide range of areas, since they complement traditional remote sensing data (e.g., satellite images) and offer a rapidly, precise, detail and high-resolution images that fit well for environmental studies. Herein, the use of high-resolution RPAS images for ESI analysis of rocky shores in the Brazilian territory was performed. Using RPAS images, with their higher-resolution compared with ArcGIS Pro Basemap satellite images, increased the detailed of ESI analysis for oil spills, increasing the number of regions in the rocky shore that are more sensitive to oil spills. The RPAS images were able to decrease the number of areas that were less sensitive to oil spills, and increase areas that are more sensitive to oil spills. This increase is important, since they were not detected in the ESI analysis using conventional ArcGIS Pro Basemap satellite images. The RPAS images permit to delineate precisely rocky shores, improving ESI interpretation in rocky shores.

Ebola virus-mediated T-lymphocyte depletion is the result of an abortive infection.

Ebola virus (EBOV) infections are characterized by a pronounced lymphopenia that is highly correlative with fatalities. However, the mechanisms leading to T-cell depletion remain largely unknown. Here, we demonstrate that both viral mRNAs and antigens are detectable in CD4+ T cells despite the absence of productive infection. A protein phosphatase 1 inhibitor, 1E7-03, and siRNA-mediated suppression of viral antigens were used to demonstrate de novo synthesis of viral RNAs and antigens in CD4+ T cells, respectively. Cell-to-cell fusion of permissive Huh7 cells with non-permissive Jurkat T cells impaired productive EBOV infection suggesting the presence of a cellular restriction factor. We determined that viral transcription is partially impaired in the fusion T cells. Lastly, we demonstrate that exposure of T cells to EBOV resulted in autophagy through activation of ER-stress related pathways. These data indicate that exposure of T cells to EBOV results in an abortive infection, which likely contributes to the lymphopenia observed during EBOV infections.

5,926 hysterectomies: complications described by Clavien-Dindo classification.

Hysterectomy is the most common major gynaecological surgery. Due to its high volume, the analysis of its results is relevant. The objective of this study was to describe intraoperative complications and reoperations, for both benign and malignant causes, using the Clavien-Dindo classification (approved by local ethics committee, number 100220). Between 2000 and 2019, 5926 elective hysterectomies were performed, of which 90.2% were for benign aetiology and 9.8% for malignant causes. The abdominal route was 52.7%, vaginal 40.1% and laparoscopic 7.2%. Intraoperative complications and reoperations (grade III Clavien-Dindo) were 4% and 2.1%, respectively. Oncological surgery had significantly more intraoperative complications (10% vs. 3.4%) and reoperations (3.6% vs. 1.9%) than benign procedures. Noteworthy, intraoperative complications required a new operation in only 3.4% for malignant and 2.8% for benign surgery. Our data showed the relevance of detecting and rectifying intraoperative complications during surgery, which consequently leads to a lower reoperation rate, minimising postoperative morbidity and mortality for patients.Impact StatementWhat is already known on this subject? The surgical complications of hysterectomy, both intraoperative and postoperative, are extensively described. However, this information is not well systematised, in which elective and emergency surgery are mixed. In addition to the above, there are few documents comparing the results of hysterectomies due to benign versus malignant causes.What the results of this study add? Using the Clavien-Dindo classification, this study adds an organised description of intraoperative complications and reoperations of hysterectomy in the context of elective surgery. In addition, it provides information on the comparison between surgery for benign versus malignant causes, as well as information on intraoperative complications requiring a new operation.What the implications are of these findings for clinical practice and/or further research? These findings provide clear and orderly data about the risks of elective hysterectomy and showed the relevance of detecting and rectifying intraoperative complications during the procedure. This is useful for specialists to preoperatively identify the risks for each hysterectomy group and provide their patients with more detailed information during informed consent.

Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap.

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection.

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

Ebola Virus Shed Glycoprotein Triggers Differentiation, Infection, and Death of Monocytes Through Toll-Like Receptor 4 Activation.

A better understanding of the mechanisms used by Ebola virus to disable the host immune system and spread the infection are of great importance for development of new therapeutic strategies. We demonstrate that treatment of monocytic cells with Ebola virus shed glycoprotein (GP) promotes their differentiation resulting in increased infection and cell death. The effects were inhibited by blocking Toll-like receptor 4 pathway. In addition, high levels of shed GP were detected in supernatants of cells treated with Ebola vaccines. This study highlights the role of shed GP in Ebola pathogenesis and also in adverse effects associated with Ebola vaccines.

Disruption of Phosphatidylserine Synthesis or Trafficking Reduces Infectivity of Ebola Virus.

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.

Role of Transmembrane Protein 16F in the Incorporation of Phosphatidylserine Into Budding Ebola Virus Virions.

Viral apoptotic mimicry, which is defined by exposure of phosphatidylserine (PtdSer) into the outer leaflet of budding enveloped viruses, increases viral tropism, infectivity and promotes immune evasion. Here, we report that the calcium (Ca2+)-dependent scramblase, transmembrane protein 16F (TMEM16F), is responsible for the incorporation of PtdSer into virion membranes during Ebola virus infection. Infection of Huh7 cells with Ebola virus resulted in a pronounced increase in plasma membrane-associated PtdSer, which was demonstrated to be dependent on TMEM16F function. Analysis of virions using imaging flow cytometry revealed that short hairpin RNA-mediated down-regulation of TMEM16F function directly reduced virion-associated PtdSer. Taken together, these studies demonstrate that TMEM16F is a central cellular factor in the exposure of PtdSer in the outer leaflet of viral membranes.

The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses.

Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4+ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4+ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt EBOV. These results demonstrate a global suppression of cell-mediated responses by EBOV IIDs and identify the role of DCs in suppression of T-cell responses.

Highly fermentable starch at different diet starch concentrations decreased feed intake and milk yield of cows in the early postpartum period.

The objective of this study was to evaluate the effects of diet starch concentration and fermentability (SF) fed during the early postpartum (PP) period on dry matter intake (DMI), yields of milk and milk components, body reserves, and metabolism. Fifty-two multiparous Holstein cows were used in a randomized block design with a 2 × 2 factorial arrangement of treatments. Treatment diets were formulated to 22% (LS) or 28% (HS) starch with dry ground corn (DGC) or high-moisture corn (HMC) as the primary starch source. Treatments were fed from 1 to 23 d PP and cows were switched to a common diet until 72 d PP to measure carryover (CO) effects. Treatment period (TP) diets were formulated for 22% forage neutral detergent fiber and 17% crude protein, and starch concentration was adjusted by substitution of corn grain for soyhulls. Throughout the experiment DMI and milk yield were measured daily, and milk components, body condition score (BCS), and body weight were measured weekly. Blood was collected weekly during the TP and every second week during the CO period. During the TP, HMC decreased DMI more when included in the HS (3.9 kg/d) than in the LS (0.9 kg/d) diets and HMC decreased yields of milk, fat, and FCM by 4.3, 0.19, and 4.8 kg/d, respectively. Treatments also interacted over time to decrease DMI and yields of milk and milk components more for HMC compared with DGC as time progressed during the TP. Loss of BCS was increased when HMC was fed in a HS diet (-0.38 vs. -0.17) and decreased when included in a LS diet (-0.21 vs. -0.29) with no effects on body weight change during the TP. Treatments interacted with time to affect plasma concentrations of glucose and insulin with HS increasing concentrations early in the TP compared with LS but with similar effects by the end of the TP. During the CO period, treatment effects on DMI diminished over time with no main effects of treatment for the entire period. Starch concentration and SF interacted to affect yields of milk, fat, and FCM during the CO period, which were greater for HS-DGC and LS-HMC (54.8 and 52.8, 1.76 and 1.81, and 51.3 and 52.2 kg/d, respectively) than for LS-DGC and HS-HMC (51.2 and 51.0, 1.68 and 1.64, and 48.4 and 48.6 kg/d, respectively). Treatments did not affect BCS change during the CO period but HS lost body weight compared with LS (-5.7 vs. 7.0 kg). Blood glucose and insulin concentrations were not affected by treatments during the CO period. Feeding a highly fermentable starch source during the early PP period decreased DMI and yields of milk and milk components compared with a less fermentable starch source and the depression in DMI was greater when fed in the higher starch diet. However, diet starch concentration had no effects on yield of milk or milk components.

ß2-Adrenergic receptor agonist ameliorates phenotypes and corrects microRNA-mediated IGF1 deficits in a mouse model of Rett syndrome.

Rett syndrome is a severe childhood onset neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein 2 (MECP2), with known disturbances in catecholamine synthesis. Here, we show that treatment with the ß2-adrenergic receptor agonist clenbuterol increases survival, rescues abnormalities in respiratory function and social recognition, and improves motor coordination in young male Mecp2-null (Mecp2(-/y)) mice. Importantly, we demonstrate that short-term treatment with clenbuterol in older symptomatic female heterozygous (Mecp2(-/+)) mice rescues respiratory, cognitive, and motor coordination deficits, and induces an anxiolytic effect. In addition, we reveal abnormalities in a microRNA-mediated pathway, downstream of brain-derived neurotrophic factor that affects insulin-like growth factor 1 (IGF1) expression in Mecp2(-/y) mice, and show that treatment with clenbuterol restores the observed molecular alterations. Finally, cotreatment with clenbuterol and recombinant human IGF1 results in additional increases in survival in male null mice. Collectively, our data support a role for IGF1 and other growth factor deficits as an underlying mechanism of Rett syndrome and introduce ß2-adrenergic receptor agonists as potential therapeutic agents for the treatment of the disorder.

Functional recovery with recombinant human IGF1 treatment in a mouse model of Rett Syndrome.

Rett Syndrome is a neurodevelopmental disorder that arises from mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2). MeCP2 has a large number of targets and a wide range of functions, suggesting the hypothesis that functional signaling mechanisms upstream of synaptic and circuit maturation may contribute to our understanding of the disorder and provide insight into potential treatment. Here, we show that insulin-like growth factor-1 (IGF1) levels are reduced in young male Mecp2-null (Mecp2(-/y)) mice, and systemic treatment with recombinant human IGF1 (rhIGF1) improves lifespan, locomotor activity, heart rate, respiration patterns, and social and anxiety behavior. Furthermore, Mecp2-null mice treated with rhIGF1 show increased synaptic and activated signaling pathway proteins, enhanced cortical excitatory synaptic transmission, and restored dendritic spine densities. IGF1 levels are also reduced in older, fully symptomatic heterozygous (Mecp2(-/+)) female mice, and short-term treatment with rhIGF1 in these animals improves respiratory patterns, reduces anxiety levels, and increases exploratory behavior. In addition, rhIGF1 treatment normalizes abnormally prolonged plasticity in visual cortex circuits of adult Mecp2(-/+) female mice. Our results provide characterization of the phenotypic development of Rett Syndrome in a mouse model at the molecular, circuit, and organismal levels and demonstrate a mechanism-based therapeutic role for rhIGF1 in treating Rett Syndrome.

Landslides Zonation Hazard: relation between geological structures and landslides occurrence in hilly tropical regions of Brazil.

This paper presents a new approach of landslides zonation hazard studies, based on an integrated study of structural data along with geomorphological and external factors, in a hilly regions of Brazil, covered by a tropical humid rain-forest, called Serra do Mar. The Serra do Mar consists of a hilly region along the east coast of Brazil, with high slopes and many geological structures in a gneiss - migmatitic terrain. In contrast to traditional approaches, this method proposes that structural data (foliation, fractures and bedding planes) and its relation with the slope geometry, is important to be consider in the landslide zonation hazard, along with declivity, relative relief, soil and rock properties, land use and vegetation cover and hydrogeological and climate factors. Results show that slopes with high hazard have the same dip direction of geological structures. Landslide zonation hazard using structural data contributes to a better understanding of how these structures, preserved in tropical residual soils, influence on slope stability and generates landslides.

CT Dose Optimization in Pediatric Radiology: A Multiyear Effort to Preserve the Benefits of Imaging While Reducing the Risks.

The marked increase in radiation exposure from medical imaging, especially in children, has caused considerable alarm and spurred efforts to preserve the benefits but reduce the risks of imaging. Applying the principles of the Image Gently campaign, data-driven process and quality improvement techniques such as process mapping and flowcharting, cause-and-effect diagrams, Pareto analysis, statistical process control (control charts), failure mode and effects analysis, "lean" or Six Sigma methodology, and closed feedback loops led to a multiyear program that has reduced overall computed tomographic (CT) examination volume by more than fourfold and concurrently decreased radiation exposure per CT study without compromising diagnostic utility. This systematic approach involving education, streamlining access to magnetic resonance imaging and ultrasonography, auditing with comparison with benchmarks, applying modern CT technology, and revising CT protocols has led to a more than twofold reduction in CT radiation exposure between 2005 and 2012 for patients at the authors' institution while maintaining diagnostic utility.

Adaptive responses of mitochondria to mild copper deprivation involve changes in morphology, OXPHOS remodeling and bioenergetics.

Copper is an essential cofactor of complex IV of the electron transfer chain, and it is directly involved in the generation of mitochondrial membrane potential. Its deficiency induces the formation of ROS, large mitochondria and anemia. Thus, there is a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis. Copper depletion might end in cellular apoptosis or necrosis. However, before entering into those irreversible processes, mitochondria may execute a series of adaptive responses. Mitochondrial adaptive responses (MAR) may involve multiple and diverse mechanisms for preserving cell life, such as mitochondrial dynamics, OXPHOS remodeling and bioenergetics output. In this study, a mild copper deficiency was produced in an animal model through intraperitoneal injections of bathocuproine disulfonate in order to study the MAR. Under these conditions, a new type of mitochondrial morphology was discovered in the liver. Termed the "butternut squash" mitochondria, it coexisted with normal and swollen mitochondria. Western blot analyses of mitochondrial dynamics proteins showed an up-regulation of MFN-2 and OPA1 fusion proteins. Furthermore, isolated liver mitochondria displayed OXPHOS remodeling through a decrease in supercomplex activity with a concomitant increase at an individual level of complexes I and IV, higher respiratory rates at complex I and II levels, higher oligomycin-insensitive respiration, and lower respiratory control ratio values when compared to the control group. As expected, total ATP and ATP/ADP values were not significantly different, since animal's health was not compromised. As a whole, these results describe a compensatory and adaptive response of metabolism and bioenergetics under copper deprivation.

Functional dissection of complex and molecular trait variants at single nucleotide resolution.

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics. The majority of these variants have individually weak effects and lie in non-coding gene-regulatory elements where we lack a complete understanding of how single nucleotide alterations modulate transcriptional processes to affect human phenotypes. To address this, we measured the activity of 221,412 trait-associated variants that had been statistically fine-mapped using a Massively Parallel Reporter Assay (MPRA) in 5 diverse cell-types. We show that MPRA is able to discriminate between likely causal variants and controls, identifying 12,025 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% can plausibly be explained by the disruption of a known transcription factor (TF) binding motif. We dissect the mechanisms of 136 variants using saturation mutagenesis and assign impacted TFs for 91% of variants without a clear canonical mechanism. Finally, we provide evidence that epistasis is prevalent for variants in close proximity and identify multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants underlying complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells.

Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.