Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

The signalling pathways initiated by members of the transforming growth factor-β (TGFβ) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT) and apoptosis. TGFβ signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFβ signalling pathway, we performed a pharmacological screen by using a cell line engineered to report the endogenous transcription of the TGFβ-responsive target gene PAI-1. The screen revealed that small molecule inhibitors of salt-inducible kinases (SIKs) attenuate TGFβ-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFβ-dependent transcriptional responses. Pharmacological inhibition of SIKs by using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFβ stimulation. Our data therefore provide evidence for a novel function of SIKs in modulating TGFβ-mediated transcriptional and cellular responses.</p

Identifying and Seeing beyond Multiple Sequence Alignment Errors Using Intra-Molecular Protein Covariation

BACKGROUND: There is currently no way to verify the quality of a multiple sequence alignment that is independent of the assumptions used to build it. Sequence alignments are typically evaluated by a number of established criteria: sequence conservation, the number of aligned residues, the frequency of gaps, and the probable correct gap placement. Covariation analysis is used to find putatively important residue pairs in a sequence alignment. Different alignments of the same protein family give different results demonstrating that covariation depends on the quality of the sequence alignment. We thus hypothesized that current criteria are insufficient to build alignments for use with covariation analyses. METHODOLOGY/PRINCIPAL FINDINGS: We show that current criteria are insufficient to build alignments for use with covariation analyses as systematic sequence alignment errors are present even in hand-curated structure-based alignment datasets like those from the Conserved Domain Database. We show that current non-parametric covariation statistics are sensitive to sequence misalignments and that this sensitivity can be used to identify systematic alignment errors. We demonstrate that removing alignment errors due to 1) improper structure alignment, 2) the presence of paralogous sequences, and 3) partial or otherwise erroneous sequences, improves contact prediction by covariation analysis. Finally we describe two non-parametric covariation statistics that are less sensitive to sequence alignment errors than those described previously in the literature. CONCLUSIONS/SIGNIFICANCE: Protein alignments with errors lead to false positive and false negative conclusions (incorrect assignment of covariation and conservation, respectively). Covariation analysis can provide a verification step, independent of traditional criteria, to identify systematic misalignments in protein alignments. Two non-parametric statistics are shown to be somewhat insensitive to misalignment errors, providing increased confidence in contact prediction when analyzing alignments with erroneous regions because of an emphasis on they emphasize pairwise covariation over group covariation

Expression and Functional Studies of Ubiquitin C-Terminal Hydrolase L1 Regulated Genes

Deubiquitinating enzymes (DUBs) have been increasingly implicated in regulation of cellular processes, but a functional role for Ubiquitin C-terminal Hydrolases (UCHs), which has been largely relegated to processing of small ubiquitinated peptides, remains unexplored. One member of the UCH family, UCH L1, is expressed in a number of malignancies suggesting that this DUB might be involved in oncogenic processes, and increased expression and activity of UCH L1 have been detected in EBV-immortalized cell lines. Here we present an analysis of genes regulated by UCH L1 shown by microarray profiles obtained from cells in which expression of the gene was inhibited by RNAi. Microarray data were verified with subsequent real-time PCR analysis. We found that inhibition of UCH L1 activates genes that control apoptosis, cell cycle arrest and at the same time suppresses expression of genes involved in proliferation and migration pathways. These findings are complemented by biological assays for apoptosis, cell cycle progression and migration that support the data obtained from microarray analysis, and suggest that the multi-functional molecule UCH L1 plays a role in regulating principal pathways involved in oncogenesis

Identification of Coevolving Residues and Coevolution Potentials Emphasizing Structure, Bond Formation and Catalytic Coordination in Protein Evolution

The structure and function of a protein is dependent on coordinated interactions between its residues. The selective pressures associated with a mutation at one site should therefore depend on the amino acid identity of interacting sites. Mutual information has previously been applied to multiple sequence alignments as a means of detecting coevolutionary interactions. Here, we introduce a refinement of the mutual information method that: 1) removes a significant, non-coevolutionary bias and 2) accounts for heteroscedasticity. Using a large, non-overlapping database of protein alignments, we demonstrate that predicted coevolving residue-pairs tend to lie in close physical proximity. We introduce coevolution potentials as a novel measure of the propensity for the 20 amino acids to pair amongst predicted coevolutionary interactions. Ionic, hydrogen, and disulfide bond-forming pairs exhibited the highest potentials. Finally, we demonstrate that pairs of catalytic residues have a significantly increased likelihood to be identified as coevolving. These correlations to distinct protein features verify the accuracy of our algorithm and are consistent with a model of coevolution in which selective pressures towards preserving residue interactions act to shape the mutational landscape of a protein by restricting the set of admissible neutral mutations

Changes in muscle contractile characteristics and jump height following 24 days of unilateral lower limb suspension

We measured changes in maximal voluntary and electrically evoked torque and rate of torque development because of limb unloading. We investigated whether these changes during single joint isometric muscle contractions were related to changes in jump performance involving dynamic muscle contractions and several joints. Six healthy male subjects (21 ± 1 years) underwent 3 weeks of unilateral lower limb suspension (ULLS) of the right limb. Plantar flexor and knee extensor maximal voluntary contraction (MVC) torque and maximal rate of torque development (MRTD), voluntary activation, and maximal triplet torque (thigh; 3 pulses at 300 Hz) were measured next to squat jump height before and after ULLS. MVC of plantar flexors and knee extensors (MVCke) and triplet torque decreased by 12% (P = 0.012), 21% (P = 0.001) and 11% (P = 0.016), respectively. Voluntary activation did not change (P = 0.192). Absolute MRTD during voluntary contractions decreased for plantar flexors (by 17%, P = 0.027) but not for knee extensors (P = 0.154). Absolute triplet MRTD decreased by 17% (P = 0.048). The reduction in MRTD disappeared following normalization to MVC. Jump height with the previously unloaded leg decreased significantly by 28%. No significant relationships were found between any muscle variable and jump height (r < 0.48), but decreases in torque were (triplet, r = 0.83, P = 0.04) or tended to be (MVCke r = 0.71, P = 0.11) related to decreases in jump height. Thus, reductions in isometric muscle torque following 3 weeks of limb unloading were significantly related to decreases in the more complex jump task, although torque in itself (without intervention) was not related to jump performance

A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues

Integrated Analysis of Residue Coevolution and Protein Structure in ABC Transporters

Intraprotein side chain contacts can couple the evolutionary process of amino acid substitution at one position to that at another. This coupling, known as residue coevolution, may vary in strength. Conserved contacts thus not only define 3-dimensional protein structure, but also indicate which residue-residue interactions are crucial to a protein’s function. Therefore, prediction of strongly coevolving residue-pairs helps clarify molecular mechanisms underlying function. Previously, various coevolution detectors have been employed separately to predict these pairs purely from multiple sequence alignments, while disregarding available structural information. This study introduces an integrative framework that improves the accuracy of such predictions, relative to previous approaches, by combining multiple coevolution detectors and incorporating structural contact information. This framework is applied to the ABC-B and ABC-C transporter families, which include the drug exporter P-glycoprotein involved in multidrug resistance of cancer cells, as well as the CFTR chloride channel linked to cystic fibrosis disease. The predicted coevolving pairs are further analyzed based on conformational changes inferred from outward- and inward-facing transporter structures. The analysis suggests that some pairs coevolved to directly regulate conformational changes of the alternating-access transport mechanism, while others to stabilize rigid-body-like components of the protein structure. Moreover, some identified pairs correspond to residues previously implicated in cystic fibrosis

Maternal Dietary Supplementation with Oligofructose-Enriched Inulin in Gestating/Lactating Rats Preserves Maternal Bone and Improves Bone Microarchitecture in Their Offspring

This study received financial support from Abbott Nutrition, a commercial company, and coauthors PBV, MM, JMLP and RR are employees of Abbott Nutrition. There are two patents related with the data presented (EP 2502507 A1 and EP 2745706 A1).Some of these results were presented in the 7th World Congress of DOHaD (2011) and in the World Congress on Osteoporosis, Osteoarthritis and Musculoskeletal Disease (WCO-IOF-ESCEO) (2014).Nutrition during pregnancy and lactation could exert a key role not only on maternal bone, but also could influence the skeletal development of the offspring. This study was performed in rats to assess the relationship between maternal dietary intake of prebiotic oligofructose-enriched inulin and its role in bone turnover during gestation and lactation, as well as its effect on offspring peak bone mass/architecture during early adulthood. Rat dams were fed either with standard rodent diet (CC group), calcium-fortified diet (Ca group), or prebiotic oligofructose-enriched inulin supplemented diet (Pre group), during the second half of gestation and lactation. Bone mineral density (BMD) and content (BMC), as well as micro-structure of dams and offspring at different stages were analysed. Dams in the Pre group had significantly higher trabecular thickness (Tb.Th), trabecular bone volume fraction (BV/TV) and smaller specific bone surface (BS/BV) of the tibia in comparison with CC dams. The Pre group offspring during early adulthood had an increase of the lumbar vertebra BMD when compared with offspring of CC and Ca groups. The Pre group offspring also showed significant increase versus CC in cancellous and cortical structural parameters of the lumbar vertebra 4 such as Tb.Th, cortical BMD and decreased BS/BV. The results indicate that oligofructose-enriched inulin supplementation can be considered as a plausible nutritional option for protecting against maternal bone loss during gestation and lactation preventing bone fragility and for optimizing peak bone mass and architecture of the offspring in order to increase bone strength.This study was funded by Abbott Nutrition R&D, and co-authors PBV, MM, JMLP and RR receive salary from Abbott Nutrition

Measuring progress from 1990 to 2017 and projecting attainment to 2030 of the health-related Sustainable Development Goals for 195 countries and territories: a systematic analysis for the Global Burden of Disease Study 2017

Background Efforts to establish the 2015 baseline and monitor early implementation of the UN Sustainable Development Goals (SDGs) highlight both great potential for and threats to improving health by 2030. To fully deliver on the SDG aim of “leaving no one behind”, it is increasingly important to examine the health-related SDGs beyond national-level estimates. As part of the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017), we measured progress on 41 of 52 health-related SDG indicators and estimated the health-related SDG index for 195 countries and territories for the period 1990–2017, projected indicators to 2030, and analysed global attainment. Methods We measured progress on 41 health-related SDG indicators from 1990 to 2017, an increase of four indicators since GBD 2016 (new indicators were health worker density, sexual violence by non-intimate partners, population census status, and prevalence of physical and sexual violence [reported separately]). We also improved the measurement of several previously reported indicators. We constructed national-level estimates and, for a subset of health-related SDGs, examined indicator-level differences by sex and Socio-demographic Index (SDI) quintile. We also did subnational assessments of performance for selected countries. To construct the healthrelated SDG index, we transformed the value for each indicator on a scale of 0–100, with 0 as the 2·5th percentile and 100 as the 97·5th percentile of 1000 draws calculated from 1990 to 2030, and took the geometric mean of the scaled indicators by target. To generate projections through 2030, we used a forecasting framework that drew estimates from the broader GBD study and used weighted averages of indicator-specific and country-specific annualised rates of change from 1990 to 2017 to inform future estimates. We assessed attainment of indicators with defined targets in two ways: first, using mean values projected for 2030, and then using the probability of attainment in 2030 calculated from 1000 draws. We also did a global attainment analysis of the feasibility of attaining SDG targets on the basis of past trends. Using 2015 global averages of indicators with defined SDG targets, we calculated the global annualised rates of change required from 2015 to 2030 to meet these targets, and then identified in what percentiles the required global annualised rates of change fell in the distribution of country-level rates of change from 1990 to 2015. We took the mean of these global percentile values across indicators and applied the past rate of change at this mean global percentile to all health-related SDG indicators, irrespective of target definition, to estimate the equivalent 2030 global average value and percentage change from 2015 to 2030 for each indicator. Findings The global median health-related SDG index in 2017 was 59·4 (IQR 35·4–67·3), ranging from a low of 11·6 (95% uncertainty interval 9·6–14·0) to a high of 84·9 (83·1–86·7). SDG index values in countries assessed at the subnational level varied substantially, particularly in China and India, although scores in Japan and the UK were more homogeneous. Indicators also varied by SDI quintile and sex, with males having worse outcomes than females for non-communicable disease (NCD) mortality, alcohol use, and smoking, among others. Most countries were projected to have a higher health-related SDG index in 2030 than in 2017, while country-level probabilities of attainment by 2030 varied widely by indicator. Under-5 mortality, neonatal mortality, maternal mortality ratio, and malaria indicators had the most countries with at least 95% probability of target attainment. Other indicators, including NCD mortality and suicide mortality, had no countries projected to meet corresponding SDG targets on the basis of projected mean values for 2030 but showed some probability of attainment by 2030. For some indicators, including child malnutrition, several infectious diseases, and most violence measures, the annualised rates of change required to meet SDG targets far exceeded the pace of progress achieved by any country in the recent past. We found that applying the mean global annualised rate of change to indicators without defined targets would equate to about 19% and 22% reductions in global smoking and alcohol consumption, respectively; a 47% decline in adolescent birth rates; and a more than 85% increase in health worker density per 1000 population by 2030. Interpretation The GBD study offers a unique, robust platform for monitoring the health-related SDGs across demographic and geographic dimensions. Our findings underscore the importance of increased collection and analysis of disaggregated data and highlight where more deliberate design or targeting of interventions could accelerate progress in attaining the SDGs. Current projections show that many health-related SDG indicators, NCDs, NCD-related risks, and violence-related indicators will require a concerted shift away from what might have driven past gains—curative interventions in the case of NCDs—towards multisectoral, prevention-oriented policy action and investments to achieve SDG aims. Notably, several targets, if they are to be met by 2030, demand a pace of progress that no country has achieved in the recent past. The future is fundamentally uncertain, and no model can fully predict what breakthroughs or events might alter the course of the SDGs. What is clear is that our actions—or inaction—today will ultimately dictate how close the world, collectively, can get to leaving no one behind by 2030

Measuring progress from 1990 to 2017 and projecting attainment to 2030 of the health-related Sustainable Development Goals for 195 countries and territories: a systematic analysis for the Global Burden of Disease Study 2017

Background: Efforts to establish the 2015 baseline and monitor early implementation of the UN Sustainable Development Goals (SDGs) highlight both great potential for and threats to improving health by 2030. To fully deliver on the SDG aim of “leaving no one behind”, it is increasingly important to examine the health-related SDGs beyond national-level estimates. As part of the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017), we measured progress on 41 of 52 health-related SDG indicators and estimated the health-related SDG index for 195 countries and territories for the period 1990–2017, projected indicators to 2030, and analysed global attainment. Methods: We measured progress on 41 health-related SDG indicators from 1990 to 2017, an increase of four indicators since GBD 2016 (new indicators were health worker density, sexual violence by non-intimate partners, population census status, and prevalence of physical and sexual violence [reported separately]). We also improved the measurement of several previously reported indicators. We constructed national-level estimates and, for a subset of health-related SDGs, examined indicator-level differences by sex and Socio-demographic Index (SDI) quintile. We also did subnational assessments of performance for selected countries. To construct the health-related SDG index, we transformed the value for each indicator on a scale of 0–100, with 0 as the 2·5th percentile and 100 as the 97·5th percentile of 1000 draws calculated from 1990 to 2030, and took the geometric mean of the scaled indicators by target. To generate projections through 2030, we used a forecasting framework that drew estimates from the broader GBD study and used weighted averages of indicator-specific and country-specific annualised rates of change from 1990 to 2017 to inform future estimates. We assessed attainment of indicators with defined targets in two ways: first, using mean values projected for 2030, and then using the probability of attainment in 2030 calculated from 1000 draws. We also did a global attainment analysis of the feasibility of attaining SDG targets on the basis of past trends. Using 2015 global averages of indicators with defined SDG targets, we calculated the global annualised rates of change required from 2015 to 2030 to meet these targets, and then identified in what percentiles the required global annualised rates of change fell in the distribution of country-level rates of change from 1990 to 2015. We took the mean of these global percentile values across indicators and applied the past rate of change at this mean global percentile to all health-related SDG indicators, irrespective of target definition, to estimate the equivalent 2030 global average value and percentage change from 2015 to 2030 for each indicator. Findings: The global median health-related SDG index in 2017 was 59·4 (IQR 35·4–67·3), ranging from a low of 11·6 (95% uncertainty interval 9·6–14·0) to a high of 84·9 (83·1–86·7). SDG index values in countries assessed at the subnational level varied substantially, particularly in China and India, although scores in Japan and the UK were more homogeneous. Indicators also varied by SDI quintile and sex, with males having worse outcomes than females for non-communicable disease (NCD) mortality, alcohol use, and smoking, among others. Most countries were projected to have a higher health-related SDG index in 2030 than in 2017, while country-level probabilities of attainment by 2030 varied widely by indicator. Under-5 mortality, neonatal mortality, maternal mortality ratio, and malaria indicators had the most countries with at least 95% probability of target attainment. Other indicators, including NCD mortality and suicide mortality, had no countries projected to meet corresponding SDG targets on the basis of projected mean values for 2030 but showed some probability of attainment by 2030. For some indicators, including child malnutrition, several infectious diseases, and most violence measures, the annualised rates of change required to meet SDG targets far exceeded the pace of progress achieved by any country in the recent past. We found that applying the mean global annualised rate of change to indicators without defined targets would equate to about 19% and 22% reductions in global smoking and alcohol consumption, respectively; a 47% decline in adolescent birth rates; and a more than 85% increase in health worker density per 1000 population by 2030. Interpretation: The GBD study offers a unique, robust platform for monitoring the health-related SDGs across demographic and geographic dimensions. Our findings underscore the importance of increased collection and analysis of disaggregated data and highlight where more deliberate design or targeting of interventions could accelerate progress in attaining the SDGs. Current projections show that many health-related SDG indicators, NCDs, NCD-related risks, and violence-related indicators will require a concerted shift away from what might have driven past gains—curative interventions in the case of NCDs—towards multisectoral, prevention-oriented policy action and investments to achieve SDG aims. Notably, several targets, if they are to be met by 2030, demand a pace of progress that no country has achieved in the recent past. The future is fundamentally uncertain, and no model can fully predict what breakthroughs or events might alter the course of the SDGs. What is clear is that our actions—or inaction—today will ultimately dictate how close the world, collectively, can get to leaving no one behind by 2030