Liver RBFOX2 regulates cholesterol homeostasis via Scarb1 alternative splicing in mice

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases

Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-To-β-strand and β-strand-To-α-helix transitions and (ii) an "open-To-closed"motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-To-closed"motion, with the β-phosphate group providing the high-Affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-To-closed"motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors

A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome

Background: b2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of b2GPI generated by anti-b2GPI antibodies is pathologically important, in contrast to monomeric b2GPI which is abundant in plasma. Principal Findings: We created a dimeric inhibitor, A1-A1, to selectively target b2GPI in b2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of b2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of b2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of b2GPI present in human serum, b2GPI purified from human plasma and the individual domain V of b2GPI. We demonstrated that when b2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of b2GPI to cardiolipin, regardless of the source of b2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of b2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-b2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric b2GPI to cardiolipin. Conclusions: Our results suggest that the approach of using a dimeric inhibitor to block b2GPI in the pathologica

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Impact and regeneration mechanism after invasion by Pteridium arachnoideum (Kaulf.) Maxon (Dennstaedtiaceae) in savanna woodland areas, southwestern São Paulo State

As samambaias do gênero Pteridium caracterizam-se pelas largas frondes e por um sistema de rizomas subterrâneos que possibilita sua rápida expansão. Apresentam elevada produção de biomassa ao longo do ano, que aumenta a incidência de incêndios, e ainda liberam substâncias químicas consideradas alelopáticas. Atualmente, são conhecidas como plantas-problema, pois vêm se expandindo e ocupando grandes áreas em diversas partes do mundo, causando impacto econômico e ecológico. Nesse contexto, este estudo teve por objetivo geral a compreensão do impacto da invasão por Pteridium arachnoideum sobre a vegetação de cerrado e a elucidação dos mecanismos por meio dos quais vem sendo lentamente revertido o processo de invasão na área de estudo. Para tanto, analisamos áreas de cerradão invadidas há mais de quatro décadas na Estação Ecológica de Assis (EEcA), no sudoeste do estado de São Paulo. Analisamos a composição florística e estrutura da comunidade em uma área invadida dentro da EEcA. Avaliamos os componentes do estrato arbóreo e regenerante da comunidade, a fim de verificar quais as espécies que conseguiram ultrapassar as barreiras físicas e químicas impostas pela presença da samambaia. Para tanto, comparamos a vegetação da área invadida com uma área adjacente não invadida, com ambiente e histórico de perturbações semelhantes. Utilizamos 100 pontos quadrantes para amostragem da composição florística e, para caracterização da estrutura da comunidade, alocamos 15 parcelas de 10 m x 30 m para amostrar o estrato superior, dentro das quais foram instaladas sub-parcelas de 2 m x 30 m para o estrato intermediário e de 2 m x 2 m para o estrato inferior. Medimos a cobertura das copas das árvores dentro das parcelas e estimamos a porcentagem de cobertura do solo por Pteridium. As espécies que conseguiram ocupar a área no início da invasão são tolerantes à sombra e com ritmo de crescimento rápido, características que possibilitaram que tais espécies ultrapassassem a barreira de samambaia e se estabelecessem na área. Já as espécies do estrato inferior se assemelham às da área não invadida, indicando que está havendo uma substituição das espécies na comunidade vegetal. A baixa densidade das árvores na área invadida diminui a competição entre os indivíduos, que, por sua vez, investem em aumento de tamanho, sendo consideravelmente maiores do que as árvores do cerradão não invadido. Essas árvores, que formam o estrato arbóreo atualmente, estão facilitando a regeneração natural de espécies arbóreas nativas e, assim, revertendo, lentamente, o processo de invasão. Testamos quais variáveis, relacionadas às funções ecológicas e aspectos estruturais das árvores de grande porte que se estabeleceram nas áreas invadidas, estão favorecendo as plantas em regeneração sob suas copas. Para tanto, amostramos os indivíduos maiores que 50 cm sob a copa de árvores sistematicamente selecionadas, segundo atributos funcionais e estruturais. Verificamos que as árvores que possuem copas mais densas e perenifólias apresentaram mais regenerantes sob suas copas. A resiliência característica do cerrado tem possibilitado que as áreas invadidas consigam se recuperar, podendo se assemelhar futuramente às áreas de cerradão da EEcA, porém muito lentamente. Ações de manejo que almejam a restauração dessas áreas mais rapidamente devem focar seus esforços na eliminação do Pteridium e/ou na introdução de espécies que possuam as características das árvores que atuam como facilitadoras da regeneração.The genus Pteridium comprises bracken species recognized by large fronds and a rhizome system that allows their fast expansion. Bracken presents an elevated biomass production throughout the year, that increases fire occurrence and release chemical compounds that are considered allelopathic. Nowadays, species of the genus Pteridium are well known as problem-species, due to their expansion and occupation of large areas around the world, causing great economic and ecological impacts. Aiming at the comprehension of bracken invasion over cerrado vegetation, we studied its impact over the plant community and, also, the slow natural regeneration process of cerrado recovery after invasion. We analyzed savanna woodland areas that have been invaded for over four decades, at Assis Ecological Station (EEcA), in southwestern São Paulo State. We analyzed the floristic composition and the plant community structure in an invaded area, compared to a non invaded patch of savanna vegetation. Components of the arboreal and regeneration strata were analyzed, in order to verify which species managed to pass by the physical and chemical barrier imposed by bracken. We assessed 100 points to assemble the floristic composition, using the quarter method, and for the community structure we used 15 plots of 10 m x 30 m to assemble the superior strata. The intermediate layer was assessed by sub-plots of 2 m x 30 m and the inferior strata using 2 m x 2 m sub-plots. We measured the canopy cover and also bracken cover inside the plots. The arboreal species first occupying the invaded area are shadow tolerant and fast growing, attributes which allowed them to overtake the bracken layer and establish. Nowadays, the invasive species has been replaced by a high number of arboreal species, the inferior strata being floristically similar to the area not invaded. The low density of adult trees in the invaded area reduce the competition among individuals that end up investing in growth, with greater size compared to those trees in the non invaded area. These large trees function as nurse trees, facilitating other species to establish under their canopies. We tested which variables related to the ecological functions and tree structure are favouring the understory, in order to comprehend the ecological processes and interactions that regulate the assembly rules of the invaded community being recovered. We assembled all individuals from arboreal species over 50 cm high growing under the projection of nurse trees canopy. These nurse trees were systematically selected, for the basis of functional and structural attributes. Understory density and richness were higher under trees with dense and permanent shadow, and that was the attribute better explaining the recovery process. The high resilience of savanna have helped the recovery of invaded areas. This process, however, has been very slow. Interventions whiling to restore those areas should be focused on bracken eradication and/or introduction of those species that can facilitate the natural regeneration

Potencial of pure plantations of native species to catalyse the recovery of biodiversity and provide sustainable use of Legal Reserve

A legislação ambiental brasileira obriga a maioria das propriedades rurais a ter Áreas de Preservação Permanente (APPs), que são áreas naturais protegidas devido à fragilidade física e ecológica, e Reserva Legal (RL), cuja função é a conservação da biodiversidade e a exploração sustentável. Em vista do passivo ambiental nas propriedades rurais brasileiras, no que se refere à RL, há uma demanda por estudos que buscam discutir estratégias para a recuperação dessas áreas. A possibilidade de exploração da RL gera uma expectativa de retorno econômico, ao mesmo tempo que essas áreas prestarão serviços ecossistêmicos importantes para a sociedade. Avaliações da regeneração natural sob plantios florestais homogêneos têm demonstrado que florestas plantadas para fins comerciais podem atuar como catalisadoras da regeneração natural a depender do manejo. Contudo, poucos estudos avaliaram a regeneração sob plantios puros de espécies nativas em regiões tropicais. Nesse contexto, para dar suporte à seleção de modelos que possam ser implementados na RL, em áreas cuja vegetação original seja de fisionomia florestal, testamos o \"modelo de mosaico\", que seria composto por pequenos talhões puros. Para tanto, utilizamos parâmetros de estrutura, riqueza, diversidade funcional e potencial econômico para avaliar plantios homogêneos (PH) de espécies nativas. No capítulo 1 comparamos PH com i) plantios mistos de espécies nativas e ii) com florestas nativas de referência em diferentes graus de conservação, visando avaliar se os PHs podem, em longo prazo, promover a conservação de biodiversidade e manutenção de processos ecológicos. No capítulo 2 avaliamos se a sucessão ecológica em PH é condicionada pelos atributos da espécie plantada ou por atributos abióticos, para identificar fatores que possam limitar o sucesso desses plantios. No capítulo 3 avaliamos se a exploração sustentável de madeira em RL a partir de PHs, que não foram manejados, é uma atividade economicamente viável frente a outros tipos de investimentos de longo prazo e outras opções de compensação de RL. Do ponto de vista ecológico, verificamos que PH com espécies nativas, que não sofreram manejo, podem, em longo prazo, promover a restauração das comunidades vegetais características de florestas semideciduais, tanto no aspecto estrutural, quanto na biodiversidade e funcionalidade. Os plantios homogêneos estudados apresentaram valores semelhantes para esses atributos ecológicos em relação a florestas de referência, inclusive às florestas maduras, que seriam a meta ideal de restauração. Em longo prazo, a estrutura (densidade e área basal) e o nível de riqueza das comunidades de plantas lenhosas sob esses plantios são muito pouco influenciados por filtros bióticos ou abióticos. O ritmo de crescimento da espécie plantada foi o único atributo que explicou parcialmente a riqueza e a densidade de indivíduos adultos nos plantios, embora não tenha exercido influência sobre a composição de espécies na comunidade em regeneração. Do ponto de vista econômico, verificamos que PHs de espécies nativas que não tiveram tratos silviculturais ao longo do seu desenvolvimento apresentaram valores para a taxa interna de retorno inferiores à poupança e valor presente líquido negativos. A compensação da RL por meio de arrendamento foi mais vantajosa do que a recomposição, mesmo considerando a venda de madeira. Destacaram-se os plantios de Handroanthus heptaphyllus, Myracrodruon urundeuva e Anadenanthera colubrina var. colubrina, cujo rendimento conseguiu superar os custos da recomposição, mesmo sem manejo adequado. Este estudo demonstrou que plantios de espécies nativas, ainda que homogêneos, podem ser utilizados para fins de recomposição de reserva legal, como proposto no \"modelo de mosaico\", pois conseguem recuperar a biodiversidade e garantir a funcionalidade dos ecossistemas em longo prazo, ainda que a composição de espécies seja distinta de florestas maduras, como tem sido observado também em plantios heterogêneos. A exploração da madeira, mesmo em áreas não manejadas, contribui para o aumento da rentabilidade de propriedades de pecuária e agricultura em longo prazo. A incorporação de tratos silviculturais adequados pode impulsionar a rentabilidade dessa atividade na RL, desde que sejam respeitadas as especificidades de exploração na RL, de modo a conciliar a produção de madeira com a conservação da biodiversidade.Environmental legislation in Brazil states that most properties must have Permanently Protected Areas (PPA), which are natural protected areas due to its physical and ecological frailty, and also Legal Reserves (LR), aiming at biodiversity conservation and sustainable management. There is a huge demand for studies on strategies to recover and to manage LR due to environmental liability on rural private properties. Sustainable timber management is allowed in LR, and this may, potentially, provide economic return to the stakeholders, in addition to important ecosystem services to society as a whole. Reviews of natural regeneration under pure forest plantations have shown that planted forests can catalyse natural regeneration. However, few studies have evaluated the regeneration under pure stands of native species in tropical regions. In this context, aiming to support the selection of models that may be implemented in LR in areas which forest was the original vegetation, we tested the \"mosaic model\", which is comprised of small pure stands. We analysed the structure, richness, functional diversity and economical potential of homogeneous stands (HS) of native species. In chapter 1 we compared HSs with i) mixed plantations of native species and ii) reference ecosystems with distinct conservation status, aiming to verifiy if HS can, in the long term, foster biodiversity conservation and sustain ecological processes. In chapter 2 we analysed if ecological succession in HS is constrained by the species planted traits or by abiotic traits, in order to identify which attributes could restrict this type of planting in order to achieve the LR purposes. In chapter 3 we evaluated if sustainable timber exploitation from homogeneous stands, without silviculture practices, in LR is a viable economical activity in comparison with other types of long term investiments and other options for LR compensation. In the long term, from the ecological perspective, homogeneous stands with native species, without management except for the seedlings cultivation during three years after planting, can foster the restoration of seasonal forest plant communities in terms of structure, biodiversity and functionality. The homogeneous stands analysed had similar values for those ecological attributes when compared with reference ecosystems, including pristine forests which is the ultimate goal of restoration projects. In the long term, structure (density and basal area) and woody species richness under these stands are barely influenced by biotic or abiotic filters. Growth rate of the species planted was the only attribute that explained, in part, the richness and density of adult individuals in the stands, although it had no influence over species composition. From the economic perspective, homogeneous stands of native species without silviculture practices throughout their development had internal rates of return lower than saving and negative net present values. Compensation of LR through leasing was most profitable for the property revenue than restoration, even when timber exploitaition was included in the property income. Significant yields were assessed at stands of Handroanthus heptaphyllus, Myracrodruon urundeuva and Anadenanthera colubrina var colubrina, that exceeded the restoration costs, without any silviculture practices. This study demonstrates that native species plantations, even with only a single species, can be considered for the restoration of legal reserve, such as the \"chessboard model\", since they can foster biodiversity recovery and assure ecosystem functioning in the long term, even though species composition are distinct from pristine forests, which has been observed also in mixed plantations. In addition, timber exploitation, even in unmanaged areas, contributes for agriculture and livestock properties revenue in the long term. The incorporation of silviculture practices can bolster timber exploitation yield in the LR, as long as it considers the specificities for this activity in LR, in order to balance timber production with biodiversity conservation

Invasion impact by Pteridium arachnoideum (Kaulf.) Maxon (Dennstaedtiaceae) on a neotropical savanna

Whether management intervention is required to control biological invasions depends primarily on demonstrating species losses resulting from such invasions. Brackens of the Pteridium genus are currently regarded as a problem species that act as important ecological filters in the assembly of invaded communities. We investigated the effects of Pteridium arachnoideum invasion on the diversity, structure, floristic composition, and functional traits of cerradão in Assis, São Paulo, Brazil. We compared an invaded site with an adjacent non-invaded site. Bracken constrained the establishment of tree species, resulting in a community structure remarkably distinct from the non-invaded area. The density and basal area of the arboreal community were higher in non-invaded areas, but large trees were more frequent in the invaded areas. However, bracken did not reduce tree species diversity. Both richness and diversity were higher in the invaded area, indicating that over time, tree species richness and diversity naturally recovered, albeit slowly, in the invaded area. Therefore, one cannot attribute the loss of richness in the Cerrado vegetation to bracken invasion. Hence, we argue that, in this system, eradication of this invasive species is not likely to be cost effective, and thus, it should be a low management priority

A native ternary complex trapped in crystal reveals the catalytic mechanism of a retaining glycosyltransferase

et al.Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes including cell signaling, cell development and host-pathogen interactions. Glycosyl transfer can proceed with either ‘inversion’ or ‘retention’ of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. We report the first native ternary complex of a GT, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from Mycobacterium tuberculosis, in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate and the divalent cation cofactor, in a productive mode for catalysis. In combination with structural, chemical, enzymatic, molecular dynamics and quantum-mechanics/molecular-mechanics (QM/MM) calculations, we unravel its catalytic mechanism, providing a strong experimental support for a front-side, substrate assisted SNi-type reaction.This work was supported by the EU Contract HEALTH-F3-2011-260872, MINECO Contract BIO2013-49022-C2-2-R, and the Basque Government (to M.E.G.); MINECO Contracts CTQ2011-24292 and CTQ2014-53144-P (to J.M.LL.) and “UAB - Banco Santander Program” (to L.M.); CTQ2013-44367-C2-1-P (to P.M.) and MINECO Contract BIO2013-49022-C2-1-R (to A.P). F.G.-B. and F.M. acknowledge support from the JAE Predoc Program (CSIC) and “Becas de Doctorado en el Extranjero - Becas Chile - CONICYT” Program, respectively.Peer reviewe