Multiple Lines of Evidence Support 199 SARS-CoV-2 Positively Selected Amino Acid Sites.

SARS-CoV-2 amino acid variants that contribute to an increased transmissibility or to host immune system escape are likely to increase in frequency due to positive selection and may be identified using different methods, such as codeML, FEL, FUBAR, and MEME. Nevertheless, when using different methods, the results do not always agree. The sampling scheme used in different studies may partially explain the differences that are found, but there is also the possibility that some of the identified positively selected amino acid sites are false positives. This is especially important in the context of very large-scale projects where hundreds of analyses have been performed for the same protein-coding gene. To account for these issues, in this work, we have identified positively selected amino acid sites in SARS-CoV-2 and 15 other coronavirus species, using both codeML and FUBAR, and compared the location of such sites in the different species. Moreover, we also compared our results to those that are available in the COV2Var database and the frequency of the 10 most frequent variants and predicted protein location to identify those sites that are supported by multiple lines of evidence. Amino acid changes observed at these sites should always be of concern. The information reported for SARS-CoV-2 can also be used to identify variants of concern in other coronaviruses.

SEDA: A Desktop Tool Suite for FASTA Files Processing.

SEDA (SEquence DAtaset builder) is a multiplatform desktop application for the manipulation of FASTA files containing DNA or protein sequences. The convenient graphical user interface gives access to a collection of simple (filtering, sorting, or file reformatting, among others) and advanced (BLAST searching, protein domain annotation, gene annotation, and sequence alignment) utilities not present in similar applications, which eases the work of life science researchers working with DNA and/or protein sequences, especially those who have no programming skills. This paper presents general guidelines on how to build efficient data handling protocols using SEDA, as well as practical examples on how to prepare high-quality datasets for single gene phylogenetic studies, the characterization of protein families, or phylogenomic studies. The user-friendliness of SEDA also relies on two important features: (i) the availability of easy-to-install distributable versions and installers of SEDA, including a Docker image for Linux, and (ii) the facility with which users can manage large datasets. SEDA is open-source, with GNU General Public License v3.0 license, and publicly available at GitHub (https://github.com/sing-group/seda). SEDA installers and documentation are available at https://www.sing-group.org/seda/.

Early infection is an independent risk factor for increased mortality in patients with culture-confirmed infected pancreatic necrosis.

BACKGROUND: Mortality in infected pancreatic necrosis (IPN) is dynamic over the course of the disease, with type and timing of interventions as well as persistent organ failure being key determinants. The timing of infection onset and how it pertains to mortality is not well defined. OBJECTIVES: To determine the association between mortality and the development of early IPN. METHODS: International multicenter retrospective cohort study of patients with IPN, confirmed by a positive microbial culture from (peri) pancreatic collections. The association between timing of infection onset, timing of interventions and mortality were assessed using Cox regression analyses. RESULTS: A total of 743 patients from 19 centers across 3 continents with culture-confirmed IPN from 2000 to 2016 were evaluated, mortality rate was 20.9% (155/734). Early infection was associated with a higher mortality, when early infection occurred within the first 4 weeks from presentation with acute pancreatitis. After adjusting for comorbidity, advanced age, organ failure, enteral nutrition and parenteral nutrition, early infection (≤4 weeks) and early open surgery (≤4 weeks) were associated with increased mortality [HR: 2.45 (95% CI: 1.63-3.67), p < 0.001 and HR: 4.88 (95% CI: 1.70-13.98), p = 0.003, respectively]. There was no association between late open surgery, early or late minimally invasive surgery, early or late percutaneous drainage with mortality (p > 0.05). CONCLUSION: Early infection was associated with increased mortality, independent of interventions. Early surgery remains a strong predictor of excess mortality.

ATXN1 N-terminal region explains the binding differences of wild-type and expanded forms.

BACKGROUND: Wild-type (wt) polyglutamine (polyQ) regions are implicated in stabilization of protein-protein interactions (PPI). Pathological polyQ expansion, such as that in human Ataxin-1 (ATXN1), that causes spinocerebellar ataxia type 1 (SCA1), results in abnormal PPI. For ATXN1 a larger number of interactors has been reported for the expanded (82Q) than the wt (29Q) protein. METHODS: To understand how the expanded polyQ affects PPI, protein structures were predicted for wt and expanded ATXN1, as well as, for 71 ATXN1 interactors. Then, the binding surfaces of wt and expanded ATXN1 with the reported interactors were inferred. RESULTS: Our data supports that the polyQ expansion alters the ATXN1 conformation and that it enhances the strength of interaction with ATXN1 partners. For both ATXN1 variants, the number of residues at the predicted binding interface are greater after the polyQ, mainly due to the AXH domain. Moreover, the difference in the interaction strength of the ATXN1 variants was due to an increase in the number of interactions at the N-terminal region, before the polyQ, for the expanded form. CONCLUSIONS: There are three regions at the AXH domain that are essential for ATXN1 PPI. The N-terminal region is responsible for the strength of the PPI with the ATXN1 variants. How the predicted motifs in this region affect PPI is discussed, in the context of ATXN1 post-transcriptional modifications.

Predicting Specificities Under the Non-self Gametophytic Self-Incompatibility Recognition Model.

Non-self gametophytic self-incompatibility (GSI) recognition system is characterized by the presence of multiple F-box genes tandemly located in the S-locus, that regulate pollen specificity. This reproductive barrier is present in Solanaceae, Plantaginacea and Maleae (Rosaceae), but only in Petunia functional assays have been performed to get insight on how this recognition mechanism works. In this system, each of the encoded S-pollen proteins (called SLFs in Solanaceae and Plantaginaceae /SFBBs in Maleae) recognizes and interacts with a sub-set of non-self S-pistil proteins, called S-RNases, mediating their ubiquitination and degradation. In Petunia there are 17 SLF genes per S-haplotype, making impossible to determine experimentally each SLF specificity. Moreover, domain -swapping experiments are unlikely to be performed in large scale to determine S-pollen and S-pistil specificities. Phylogenetic analyses of the Petunia SLFs and those from two Solanum genomes, suggest that diversification of SLFs predate the two genera separation. Here we first identify putative SLF genes from nine Solanum and 10 Nicotiana genomes to determine how many gene lineages are present in the three genera, and the rate of origin of new SLF gene lineages. The use of multiple genomes per genera precludes the effect of incompleteness of the genome at the S-locus. The similar number of gene lineages in the three genera implies a comparable effective population size for these species, and number of specificities. The rate of origin of new specificities is one per 10 million years. Moreover, here we determine the amino acids positions under positive selection, those involved in SLF specificity recognition, using 10 Petunia S-haplotypes with more than 11 SLF genes. These 16 amino acid positions account for the differences of self-incompatible (SI) behavior described in the literature. When SLF and S-RNase proteins are divided according to the SI behavior, and the positively selected amino acids classified according to hydrophobicity, charge, polarity and size, we identified fixed differences between SI groups. According to the in silico 3D structure of the two proteins these amino acid positions interact. Therefore, this methodology can be used to infer SLF/S-RNase specificity recognition.

Multiple independent L-gulonolactone oxidase (GULO) gene losses and vitamin C synthesis reacquisition events in non-Deuterostomian animal species.

BACKGROUND: L-ascorbate (Vitamin C) is an important antioxidant and co-factor in eukaryotic cells, and in mammals it is indispensable for brain development and cognitive function. Vertebrates usually become L-ascorbate auxothrophs when the last enzyme of the synthetic pathway, an L-gulonolactone oxidase (GULO), is lost. Since Protostomes were until recently thought not to have a GULO gene, they were considered to be auxothrophs for Vitamin C. RESULTS: By performing phylogenetic analyses with tens of non-Bilateria and Protostomian genomes, it is shown, that a GULO gene is present in the non-Bilateria Placozoa, Myxozoa (here reported for the first time) and Anthozoa groups, and in Protostomians, in the Araneae family, the Gastropoda class, the Acari subclass (here reported for the first time), and the Priapulida, Annelida (here reported for the first time) and Brachiopoda phyla lineages. GULO is an old gene that predates the separation of Animals and Fungi, although it could be much older. We also show that within Protostomes, GULO has been lost multiple times in large taxonomic groups, namely the Pancrustacea, Nematoda, Platyhelminthes and Bivalvia groups, a pattern similar to that reported for Vertebrate species. Nevertheless, we show that Drosophila melanogaster seems to be capable of synthesizing L-ascorbate, likely through an alternative pathway, as recently reported for Caenorhabditis elegans. CONCLUSIONS: Non-Bilaterian and Protostomians seem to be able to synthesize Vitamin C either through the conventional animal pathway or an alternative pathway, but in this animal group, not being able to synthesize L-ascorbate seems to be the exception rather than the rule.

Scientific report: a case of acute pancreatitis due to liraglutide

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EvoPPI 1.0: a Web Platform for Within- and Between-Species Multiple Interactome Comparisons and Application to Nine PolyQ Proteins Determining Neurodegenerative Diseases.

Protein-protein interaction (PPI) data is essential to elucidate the complex molecular relationships in living systems, and thus understand the biological functions at cellular and systems levels. The complete map of PPIs that can occur in a living organism is called the interactome. For animals, PPI data is stored in multiple databases (e.g., BioGRID, CCSB, DroID, FlyBase, HIPPIE, HitPredict, HomoMINT, INstruct, Interactome3D, mentha, MINT, and PINA2) with different formats. This makes PPI comparisons difficult to perform, especially between species, since orthologous proteins may have different names. Moreover, there is only a partial overlap between databases, even when considering a single species. The EvoPPI ( http://evoppi.i3s.up.pt ) web application presented in this paper allows comparison of data from the different databases at the species level, or between species using a BLAST approach. We show its usefulness by performing a comparative study of the interactome of the nine polyglutamine (polyQ) disease proteins, namely androgen receptor (AR), atrophin-1 (ATN1), ataxin 1 (ATXN1), ataxin 2 (ATXN2), ataxin 3 (ATXN3), ataxin 7 (ATXN7), calcium voltage-gated channel subunit alpha1 A (CACNA1A), Huntingtin (HTT), and TATA-binding protein (TBP). Here we show that none of the human interactors of these proteins is common to all nine interactomes. Only 15 proteins are common to at least 4 of these polyQ disease proteins, and 40% of these are involved in ubiquitin protein ligase-binding function. The results obtained in this study suggest that polyQ disease proteins are involved in different functional networks. Comparisons with Mus musculus PPIs are also made for AR and TBP, using EvoPPI BLAST search approach (a unique feature of EvoPPI), with the goal of understanding why there is a significant excess of common interactors for these proteins in humans.

BDBM 1.0: A Desktop Application for Efficient Retrieval and Processing of High-Quality Sequence Data and Application to the Identification of the Putative Coffea S-Locus.

Nowadays, bioinformatics is one of the most important areas in modern biology and the creation of high-quality scientific software supporting this recent research area is one of the core activities of many researchers. In this context, high-quality sequence datasets are needed to perform inferences on the evolution of species, genes, and gene families, or to get evidence for adaptive amino acid evolution, among others. Nevertheless, sequence data are very often spread over several databases, many useful genomes and transcriptomes are non-annotated, the available annotation is not for the desired coding sequence isoform, and/or is unlikely to be accurate. Moreover, although the FASTA text-based format is quite simple and usable by most software applications, there are a number of issues that may be critical depending on the software used to analyse such files. Therefore, researchers without training in informatics often use a fraction of all available data. The above issues can be addressed using already available software applications, but there is no easy-to-use single piece of software that allows performing all these tasks within the same graphical interface, such as the one here presented, named BDBM (Blast DataBase Manager). BDBM can be used to efficiently get gene sequences from annotated and non-annotated genomes and transcriptomes. Moreover, it can be used to look for alternatives to existing annotations and to easily create reliable custom databases. Such databases are essential to prepare high-quality datasets. The analyses that we have performed on the Coffea canephora genome using BDBM aimed at the identification of the S-locus region (that harbours the genes involved in gametophytic self-incompatibility) led to the conclusion that there are two likely regions, one on chromosome 2 (around region 6600000-6650000), and another on chromosome 5 (around 15830000-15930000). Such findings are discussed in the context of the Rubiaceae gametophytic self-incompatibility evolution.

Bioinformatics Protocols for Quickly Obtaining Large-Scale Data Sets for Phylogenetic Inferences.

Useful insight into the evolution of genes and gene families can be provided by the analysis of all available genome datasets rather than just a few, which are usually those of model species. Handling and transforming such datasets into the desired format for downstream analyses is, however, often a difficult and time-consuming task for researchers without a background in informatics. Therefore, we present two simple and fast protocols for data preparation, using an easy-to-install, open-source, cross-platform software application with user-friendly, rich graphical user interface (SEDA; http://www.sing-group.org/seda/index.html ). The first protocol is a substantial improvement over one recently published (López-Fernández et al. Practical applications of computational biology and bioinformatics, 12th International conference. Springer, Cham, pp 88-96 (2019)[1]), which was used to study the evolution of GULO, a gene that encodes the enzyme responsible for the last step of vitamin C synthesis. In this paper, we show how the sequence data file used for the phylogenetic analyses can now be obtained much faster by changing the way coding sequence isoforms are removed, using the newly implemented SEDA operation "Remove isoforms". This protocol can be used to easily show that putative functional GULO genes are present in several Prostotomian groups such as Molluscs, Priapulida and Arachnida. Such findings could have been easily missed if only a few Protostomian model species had been used. The second protocol allowed us to identify positively selected amino acid sites in a set of 19 primate HLA immunity genes. Interestingly, the proteins encoded by MHC class II genes can show just as many positively selected amino acid sites as those encoded by classical MHC class I genes. Although a significant percentage of codons, which can be as high as 14.8%, are evolving under positive selection, the main mode of evolution of HLA immunity genes is purifying selection. Using a large number of primate species, the probability of missing the identification of positively selected amino acid sites is lower. Both projects were performed in less than one week, and most of the time was spent running the analyses rather than preparing the files. Such protocols can be easily adapted to answer many other questions using a phylogenetic approach.

Scientific report: a case of acute pancreatitis due to liraglutide.

Liraglutide is a long-acting glucagon-like peptide-1 (GLP-1) analog. GLP-1 analogues are used as a second option treatment for type 2 diabetes and weight management in obese patients. Data in the literature suggests an association between GLP-1 agonist use and acute pancreatitis (AP). Furthermore, it has been suggested that acute pancreatitis is a potential complication of liraglutide therapy and liraglutide should be used cautiously in patients at risk of pancreatitis. This case reported herein was a 44-year-old female diagnosed with acute pancreatitis based on 2 of 3 criteria when she presented to the Emergency Department with epigastric pain, which radiated to her back.

Influence of age, body mass index and comorbidity on major outcomes in acute pancreatitis, a prospective nation-wide multicentre study.

BACKGROUND: There are few large prospective cohort studies evaluating predictors of outcomes in acute pancreatitis. OBJECTIVES: The purpose of this study was to determine the role of age and co-morbid disease in predicting major outcomes in acute pancreatitis. METHODS: Data points were collected according to a predefined electronic data collection form. Acute pancreatitis and its complications were defined according to the revised Atlanta classification. Univariable and multivariable analyses were conducted using Cox proportional hazard regression and multiple logistic regression. RESULTS: From June 2013-February 2015, 1655 adult patients were recruited from 23 centres across Spain. Co-morbid disease, obesity, open surgical necrosectomy within 30 days, and pancreatic necrosis were independently associated with both 30-day mortality and persistent organ failure (p < 0.05 for all). Age was not associated with persistent organ failure, however the extreme of age (>85 years) was associated with mortality (p < 0.05). Co-morbid disease and obesity were not independently associated with a prolonged length of stay or other markers of morbidity on adjusted analysis (p > 0.05). CONCLUSION: Comorbidity and obesity are important determinates of mortality and persistent organ failure in acute pancreatitis, but in the absence of organ failure they do not appear to independently contribute to morbidity. This has important implications for severity classification and predictive models of severity in acute pancreatitis.

Fluid resuscitation with lactated Ringer's solution vs normal saline in acute pancreatitis: A triple-blind, randomized, controlled trial.

BACKGROUND: Little is known regarding the optimal type of fluid resuscitation in acute pancreatitis (AP). OBJECTIVE: The objective of this article was to compare the effect of lactated Ringer's solution (LR) vs normal saline (NS) in the inflammatory response in AP. METHODS: We conducted a triple-blind, randomized, controlled trial. Patients ≥ 18 admitted with AP were eligible. Patients were randomized to receive LR or NS. Primary outcome variables were number of systemic inflammatory response syndrome (SIRS) criteria at 24 hours, 48 hours and 72 hours and blood C-reactive protein (CRP) levels at 48 hours and 72 hours. In vitro complementary experiments were performed to further explore the interaction between pH, lactate and inflammation. RESULTS: Nineteen patients receiving LR and 21 receiving NS were analyzed. The median (p25-p75) number of SIRS criteria at 48 hours were 1 (1-2) for NS vs 1 (0-1) for LR, p = 0.060. CRP levels (mg/l) were as follows: at 48 hours NS 166 (78-281) vs LR 28 (3-124), p = 0.037; at 72 hours NS 217 (59-323) vs LR 25 (3-169), p = 0.043. In vitro, LR inhibited the induction of inflammatory phenotype of macrophages and NF-κB activation. This effect was not observed when using Ringer's solution without lactate, suggesting a direct anti-inflammatory effect of lactate. CONCLUSIONS: Lactated Ringer's is associated with an anti-inflammatory effect in patients with acute pancreatitis.

Large Scale Analyses and Visualization of Adaptive Amino Acid Changes Projects.

When changes at few amino acid sites are the target of selection, adaptive amino acid changes in protein sequences can be identified using maximum-likelihood methods based on models of codon substitution (such as codeml). Although such methods have been employed numerous times using a variety of different organisms, the time needed to collect the data and prepare the input files means that tens or hundreds of coding regions are usually analyzed. Nevertheless, the recent availability of flexible and easy to use computer applications that collect relevant data (such as BDBM) and infer positively selected amino acid sites (such as ADOPS), means that the entire process is easier and quicker than before. However, the lack of a batch option in ADOPS, here reported, still precludes the analysis of hundreds or thousands of sequence files. Given the interest and possibility of running such large-scale projects, we have also developed a database where ADOPS projects can be stored. Therefore, this study also presents the B+ database, which is both a data repository and a convenient interface that looks at the information contained in ADOPS projects without the need to download and unzip the corresponding ADOPS project file. The ADOPS projects available at B+ can also be downloaded, unzipped, and opened using the ADOPS graphical interface. The availability of such a database ensures results repeatability, promotes data reuse with significant savings on the time needed for preparing datasets, and effortlessly allows further exploration of the data contained in ADOPS projects.

La enteropatía de intestino delgado por antiinflamatorios no esteroideos y la hemorragia digestiva de origen oscuro / Nonsteroidal anti-inflammatory drugs-induced small bowel enteropathy and obscure gastrointestinal bleeding

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An international multicenter study of early intravenous fluid administration and outcome in acute pancreatitis.

AIMS: Early aggressive fluid resuscitation in acute pancreatitis is frequently recommended but its benefits remain unproven. The aim of this study was to determine the outcomes associated with early fluid volume administration in the emergency room (FVER) in patients with acute pancreatitis. METHODS: A four-center retrospective cohort study of 1010 patients with acute pancreatitis was conducted. FVER was defined as any fluid administered from the time of arrival to the emergency room to 4 h after diagnosis of acute pancreatitis, and was divided into tertiles: nonaggressive (<500 ml), moderate (500 to 1000 ml), and aggressive (>1000 ml). RESULTS: Two hundred sixty-nine (26.6%), 427 (42.3%), and 314 (31.1%) patients received nonaggressive, moderate, and aggressive FVER respectively. Compared with the nonaggressive fluid group, the moderate group was associated with lower rates of local complications in univariable analysis, and interventions, both in univariable and multivariable analysis (adjusted odds ratio (95% confidence interval): 0.37 (0.14-0.98)). The aggressive resuscitation group was associated with a significantly lower need for interventions, both in univariable and multivariable analysis (adjusted odds ratio 0.21 (0.05-0.84)). Increasing fluid administration categories were associated with decreasing hospital stay in univariable analysis. CONCLUSIONS: Early moderate to aggressive FVER was associated with lower need for invasive interventions.

The expression and activation of the AIM2 inflammasome correlates with inflammation and disease severity in patients with acute pancreatitis.

BACKGROUND: Acute pancreatitis is an inflammatory disorder of the pancreas that is responsible for significant morbidity and mortality. The inflammasome pathway has acquired significant relevance in the pathogenesis of many inflammatory disorders, but its role in patients with acute pancreatitis still awaits clarification. METHODS: We performed a prospective study in which 27 patients with acute pancreatitis and 16 healthy controls were included. We isolated peripheral blood mononuclear cells (PBMCs) and we assessed the expression and activation of different inflammasomes as well as their association with the clinical course of the disease. RESULTS: Our results show that PBMCs from patients with acute pancreatitis have elevated expression of several components of the inflammasome complex, including the inflammasome-forming receptor absent in melanoma 2 (AIM2), early during the onset of the disease. Activation of the AIM2 or NLRP3 inflammasomes in PBMCs from patients with acute pancreatitis results in exacerbated IL-1ß and IL-18 production compared with PBMCs from healthy controls. Furthermore, both AIM2 mRNA expression and AIM2-mediated production of IL-1ß by PBMCs correlated with increased systemic inflammation in these patients. Last, AIM2 expression was further increased in those patients that developed transient or persistent organ failure (moderate or severe acute pancreatitis). CONCLUSIONS: Our data demonstrates that AIM2 inflammasome expression and activation is increased early during the course of acute pancreatitis, and suggests that AIM2 activation may affect systemic inflammation and organ failure in these patients.

Early Predictors of Fluid Sequestration in Acute Pancreatitis: A Validation Study.

OBJECTIVES: The primary aim of this retrospective study was to externally validate predictors of increased fluid sequestration at 48 hours (FS48) in acute pancreatitis (AP). METHODS: Patients admitted between January 10 and February 13 with a diagnosis of AP were evaluated. The FS48 was calculated as difference between total fluid input and output in the first 48 hours. Predictors of FS48, such as young age, alcoholic etiology, hemoconcentration, hyperglycemia, and systemic inflammatory response syndrome (SIRS), and outcomes in AP, such as increased length of stay, acute fluid collection(s), necrosis, and persistent organ failure (POF), were defined in accordance with the previous study. Linear regression analysis was performed to evaluate the association between predictors and outcome. RESULTS: Two hundred twenty-seven AP patients (mean age, 48 years; 54% men) with a median FS48 of 4.2 L were evaluated. Age younger than 40 years, alcoholic etiology, hemoconcentration, and SIRS independently predicted increased FS48 (P < 0.05). Increased FS48 was associated with persistent SIRS and POF (P < 0.01). There was a significant trend between number of predictors and FS (P < 0.001). The presence of 4 predictors or more was associated with higher rates of persistent SIRS and POF (P < 0.01). CONCLUSIONS: Our study validated 4 of 5 predictors of increased FS48 from the previous study. Presence of 4 predictors or more and increased FS48 are both associated with persistent SIRS and POF.