Publicacións

In previous studies, we presented our site-specific Stability Constrained substitution models of Protein Evolution (Stab-CPE) that define fitness as the probability of finding a protein folded in its native state but ignore changes in the native structure. Stab-CPE models can be used to predict a more realistic evolutionary variability across protein sites, nevertheless they still qualitatively differ from observed data and appear too tolerant to mutations. Here, we present novel structurally constrained substitution models (Str-CPE) that define fitness based on the structural deformation produced by a mutation, which we predict adopting an extension of Julián Echave's linearly forced elastic network model. Compared with our previous Stab-CPE models, the novel Str-CPE models are more stringent (they predict lower sequence entropy and substitution rate), provide higher likelihood to multiple sequence alignments (MSAs) that include one or more known structures, and better predict the observed conservation across sites. The models that combine Str-CPE and Stab-CPE models are even more stringent and fit the empirical MSAs better. We collectively refer to our models as Structure and Stability Constrained substitution models of Protein Evolution (SSCPE). When using distantly related proteins, we find that more similar phylogenies are inferred under the SSCPE models than under traditional empirical substitution models if compared with the corresponding reference phylogenies inferred using structural distances. Therefore, SSCPE models seem to be much better-fitting substitution models for deep phylogeny inference. The SSCPE models have been implemented in the PERL-based program SSCPE.pl, which uses RAxML-NG to infer phylogenies under the SSCPE model given a concatenated MSA and a list of protein structures that match the sequences in the MSA. This program is freely available and downloadable from https://gihub.com/ugobas/SSCPE.

The pyrimidine ring is an important structural motif present in numerous bioactive compounds, including those with antibacterial and antitumor activities. In this study, two novel 4,6-disubstituted-2-(4-morpholinyl) pyrimidines (P12 and P14) were evaluated in breast cancer and melanoma cells with different genetic backgrounds. Both compounds demonstrated antitumor activity by reducing cell proliferation in 2D and 3D models. Mechanistic studies showed that these derivatives induce cell-cycle delay, altering the transcription of key cell-cycle mediators and leading to a senescent-like phenotype accompanied by changes in the pattern of the senescence-associated secretory phenotype (SASP). In addition, both compounds appeared to induce cell death, potentially in distinct tumor cell subpopulations. Collectively, these findings highlight the potential of pyrimidine-based derivatives as lead compounds for the development of pro-senescent anticancer agents, with utility in cancers where senescence plays a critical role, such as BRAF-mutant melanoma and the ER+/HER2- breast cancer subtype investigated in this study.

Pulmonary Arterial Hypertension (PAH) is a rare, progressive disorder characterized by pathological vascular remodeling of the pulmonary arteries, ultimately leading to right heart failure. Early diagnosis remains challenging, as clinical manifestations are often nonspecific, and definitive confirmation still requires invasive right heart catheterization. Circulating microRNAs (miRNAs) have emerged as promising biomarkers for cardiovascular diseases due to their plasma stability and direct involvement in disease pathophysiology. We performed small RNA sequencing on plasma samples from 25 IPAH patients and 10 healthy controls to identify differentially expressed miRNAs. Seven candidate miRNAs were selected for further validation by quantitative PCR in a multicenter cohort of 110 IPAH patients and controls. Logistic regression models were built to evaluate diagnostic performance. Functional studies for miR-3168 were performed using western blot and flow cytometry in Pulmonary Artery Endothelial Cells (PAECs), and tube formation assay in human umbilical vein endothelial cells (HUVECs). Our initial screen identified 29 differentially expressed miRNAs. Seven of these candidates, including members of the let-7 family and miR-3168, were successfully validated in the larger cohort. A diagnostic panel incorporating three miRNAs (let-7a-5p, miR-9-5p, and miR-31-5p) was developed, which achieved an area under the curve (AUC) of 0.862 (95% CI = 0.7481-1) for discriminating PAH patients from controls. Separately, we investigated the functional role of miR-3168, which was upregulated in PAH patients. In PAECs, overexpression of miR-3168 led to a reduction of BMPR2 protein levels. Moreover, miR-3168 overexpression impaired tube formation in HUVECs. Our study identifies a plasma-derived three-miRNA signature with strong potential for the non-invasive diagnosis of PAH. Furthermore, we implicate miR-3168 as a potential contributor to endothelial dysfunction through its regulation of BMPR2 and its anti-angiogenic effects in vitro. These findings reinforce the dual utility of circulating miRNAs as both clinically relevant non-invasive biomarkers and as tools to discover novel disease biology.

Recurrence following resection of colorectal cancer liver metastases remains a major obstacle to prolonged patient survival, often resulting in treatment-refractory disease with limited understanding of the underlying evolutionary drivers. To investigate these mechanisms, we performed an in-depth, patient-specific study of genomic and microenvironmental alterations in relapsed colorectal cancer liver metastases from nine individuals.

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of phenotypic heterogeneity. Although the predominant focus of scRNA-seq analyses has been assessing gene expression changes, several approaches have been proposed in recent years to identify changes at the DNA level from scRNA-seq data. In this study, we evaluated the relative performance of six strategies for calling single-nucleotide variants from scRNA-seq data using 381 single-cell transcriptomes from five cancer patients. Specifically, we focused on the quality of the inferred genotypes and the resulting single-cell phylogenies. We found that scAllele, Monopogen, and Monovar consistently returned phylogenetically informative genotype calls, providing more precise signals of discrimination between tumor and normal cells within heterogeneous samples and among distinct subclonal lineages in longitudinal samples. In addition, we evaluated the evolution of gene expression along the cell phylogenies. While most transcriptomic variation was very plastic and did not correlate with the cell phylogeny, a group of genes associated with cell cycle processes showed a strong phylogenetic signal in one of the patients, underscoring a potential link between gene expression patterns and lineage-specific traits in the context of cancer progression. In summary, our study highlights the potential of scRNA-seq data for inferring cell phylogenies to decipher the evolutionary dynamics of cell populations.

Substitution models of protein evolution describe the rates of evolutionary change among amino acids and are essential for a variety of evolutionary studies, including the reconstruction of phylogenetic histories and ancestral sequences, among others. The earliest substitution models of protein evolution are based on empirical protein sequences and, despite their unrealistic assumptions, are still routinely used in protein phylogenetics. Next, the incorporation of additional parameters that inform about evolutionary constraints on protein stability and protein function provided a significant increase in the accuracy of the modeling. However, despite the wide variety of substitution models of protein evolution that were presented, only a small subset has been implemented in evolutionary frameworks of practical use in phylogenetics. Here, we overview general trends in the development and application of substitution models of protein evolution, including their theoretical fundamentals, goals, areas for improvement, and implementation in phylogenetic frameworks. We also provide detailed practical examples of phylogenetic inference using advanced structurally constrained substitution models.

Human virome research is gaining increasing attention as viruses are recognized as critical modulators of microbial communities and human health. Viral metagenomics, however, faces unique challenges, including the low abundance and diversity of viruses in biological samples, the absence of universal marker genes, and biases introduced by experimental protocols. While various virome protocols have been benchmarked using viral particles or nucleic acids from mock communities, these approaches often fail to capture the complexity and heterogeneity of natural viromes. In this study, we systematically evaluated modifications to key methodological steps in the metagenomic analysis of human fecal samples, including viral enrichment, nucleic acid extraction, genome amplification, and library preparation. Using gold-standard bioinformatic approaches on sequencing datasets generated after amplification, we assessed the impact of these modifications on relative viral taxonomic assignment, contig quality, richness, diversity, and inferred genome structure. Our findings reveal striking trade-offs between recovery of viral genomes and retention of nonviral sequences, demonstrating how methodological choices can shape the inferred virome composition. Based on these observations, we propose an optimized protocol that enhances viral genome recovery while reducing contamination from nonviral sequences. This refined workflow provides a more robust and reliable framework for gut virome studies, paving the way for a deeper exploration of the role of viruses in human health and microbial ecosystems.

The genomic analysis of circulating tumor cells (CTCs) can help us identify their specific characteristics and reveal intratumor heterogeneity while also reducing the need for invasive sampling. However, studying single CTCs can be challenging, as whole-genome amplification (WGA) is needed to obtain enough genetic material for high-throughput sequencing. Here, we present a protocol for isolating single CTCs from mouse xenografts, followed by WGA using the Ampli1WGA Plus kit.

Substitution models of protein evolution describe the rates of evolutionary change among amino acids and are essential for a variety of evolutionary studies, including the reconstruction of phylogenetic histories and ancestral sequences, among others. The earliest substitution models of protein evolution are based on empirical protein sequences and, despite their unrealistic assumptions, are still routinely used in protein phylogenetics. Next, the incorporation of additional parameters that inform about evolutionary constraints on protein stability and protein function provided a significant increase in the accuracy of the modeling. However, despite the wide variety of substitution models of protein evolution that were presented, only a small subset has been implemented in evolutionary frameworks of practical use in phylogenetics. Here, we overview general trends in the development and application of substitution models of protein evolution, including their theoretical fundamentals, goals, areas for improvement, and implementation in phylogenetic frameworks. We also provide detailed practical examples of phylogenetic inference using advanced structurally constrained substitution models.

Metastatic colorectal cancer (mCRC) remains a major cause of cancer-related mortality, but few noninvasive biomarkers exist to track disease progression or inform treatment strategies. Circulating tumor cells (CTCs) offer a minimally invasive source of tumor material, yet the prognostic significance of their genomic diversity remains unclear.

The rapid spread of the invasive brown macroalga has caused severe ecological and economic damage along the European coasts. Efforts to mitigate its impact have been largely ineffective, highlighting the need for alternative strategies to valorise this invasive species. This study explores the use of aqueous extract (RO extract) as a natural reducing and stabilizing agent for the green synthesis of gold (Au@RO), silver (Ag@RO), and platinum (Pt@RO) nanoparticles. The synthesized nanoparticles were extensively characterized using ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), X-ray diffraction (XRD), zeta potential analysis, and Fourier-transform infrared spectroscopy (FTIR). The results confirmed the successful formation of spherical and stable nanoparticles. Furthermore, the antioxidant activity of the RO extract was determined before and after the synthesis of the nanoparticles by the determination of the reducing power, total phenolic content and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity. Notably, Pt@RO showed the highest enhancement in antioxidant activity among the nanoparticles synthesized. The findings demonstrate that can be repurposed as a valuable bioresource for the environmentally friendly production of metal nanoparticles with promising applications.

The primary cilium is a sensory organelle that extends from the plasma membrane. It plays a vital role in physiological and developmental processes by controlling different signalling pathways such as WNT, Sonic hedgehog (SHh), and transforming growth factor β (TGF-β). Ciliary dysfunction has been related to different pathologies such as Alström (ALMS) or Bardet-Biedl (BBS) syndrome. The leading cause of death in adults with these syndromes is chronic kidney disease (CKD), which is characterised by fibrotic and inflammatory processes often involving the TGF-β pathway. Using genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF-β signalling pathway in knockout (KO) models for ALMS1 and BBS1 genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determine which processes were deregulated in TGF-β pathway. Finally, we have analysed protein-protein and kinase-substrate interactions to prioritise candidate genes in the regulation of the TGF-β pathway in ALMS and BBS. Analysis of differentially phosphorylated proteins identified 10 candidate proteins in the ALMS1 KO model and 41 in the BBS1 KO model. After network expansion using a random walk with a restart algorithm, we were able to identify the TGF-β signalling pathway together with other related processes such as endocytosis in the case of ALMS1 or the regulation of the extracellular matrix in BBS1. Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome in ALMS1 and CDK2 in the case of BBS1. In conclusion, the depletion of ALMS1 and BBS1 affects the TGF-β signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case of ALMS1 and CDK2 in the case of BBS1.

Challenges persist regarding the influence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on public health, with growing interest in future viral molecular variants. In this context, accurate predictions demand a thorough understanding of the virus's molecular evolution, especially proteins targeted by therapies, where certain discrepancies among studies exist. We analyzed thousands of SARS-CoV-2 genomes to assess the rate of evolution and molecular adaptation in the various SARS-CoV-2 coding regions. We found an overall low genetic diversity along the genome, with fluctuations over time and among genomic regions, and a notable increase in the Omicron variant, especially in the S and ORF6 genes. We also estimated an overall rate of molecular evolution of approximately 10 substitutions per site per year, though it varied among genomic regions and over time. Actually, most genomic regions did not follow the strict molecular clock. Regarding selective pressures, the protein-coding regions of SARS-CoV-2 generally exhibited evidence of purifying selection, with local diversifying selection associated with virus transmission and replication. Overall, the molecular evolution of SARS-CoV-2 displays heterogeneity among genomic regions and over time. These findings suggest that forecasting SARS-CoV-2 molecular evolution is not straightforward and remark the importance of continuing to monitor SARS-CoV-2 evolution.

Evolutionary studies in population genetics and ecology were mainly focused on predicting and understanding past evolutionary events. Recently, however, a growing trend explores the prediction of evolutionary trajectories toward the future promoted by its wide variety of applications. In this context, we introduce a forecasting protein evolution method that integrates birth-death population models with substitution models that consider selection on protein folding stability. In contrast to traditional population genetics methods that usually make the unrealistic assumption of simulating molecular evolution separately from the evolutionary history, the present method combines both processes to simultaneously model forward-in-time birth-death evolutionary trajectories and protein evolution under structurally constrained substitution models that outperformed traditional empirical substitution models. We implemented the method into a freely available computer framework. We evaluated the accuracy of the predictions with several monitored viral proteins of broad interest. Overall, the method showed acceptable errors in predicting the folding stability of the forecasted protein variants, but, expectedly, the errors were larger in the prediction of the corresponding sequences. We conclude that forecasting protein evolution is feasible in certain evolutionary scenarios and provide suggestions to enhance its accuracy by improving the underlying models of evolution.

With rapid advancements in single-cell DNA sequencing (scDNA-seq), various computational methods have been developed to study evolution and call variants on single-cell level. However, modeling deletions remains challenging because they affect total coverage in ways that are difficult to distinguish from technical artifacts. We present DelSIEVE, a statistical method that infers cell phylogeny and single-nucleotide variants, accounting for deletions, from scDNA-seq data. DelSIEVE distinguishes deletions from mutations and artifacts, detecting more evolutionary events than previous methods. Simulations show high performance, and application to cancer samples reveals varying amounts of deletions and double mutants in different tumors.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a single-stranded, segmented RNA virus belonging to the family, and it is rapidly expanding across Africa, Asia, and southern Europe, probably favored by climate change and livestock trade. Its fatality rate in humans reaches up to 40%, and there is currently no specific treatment or vaccine available. Therefore, the development of therapies against CCHFV is essential, and their design requires understanding of the molecular evolution and genetic distribution of the virus. Motivated by these concerns, we present a comprehensive review of the molecular evolution, genetic characterization, and phylogeography of CCHFV, and we discuss their potential implications for therapeutic design. Specifically, we describe the virus's capacity to increase its genetic diversity through numerous mutations, recombination events, and genomic reassortments, which affect fundamental viral functions such as RNA binding, host-virus interactions, viral entry, and polymerase activity. We also assess the presence of temporal heterogeneous rates of evolution and molecular adaptation among CCHFV coding regions, where purifying selection is generally predominant but diversifying selection is observed in molecular regions associated with host adaptation and transmission. We emphasize the importance of understanding the complex molecular evolution of CCHFV for the rational design of therapies and highlight the need for efforts in surveillance, evolutionary prediction, and therapeutic development.

Bardet-Biedl syndrome (BBS) is a rare, genetically heterogeneous, and highly pleiotropic autosomal recessive ciliopathy. Patients typically present with early loss of vision, hyperphagia, severe obesity, learning difficulties, and renal dysfunction. In patients with BBS, dysfunction of the immotile primary cilia in the hypothalamic melanocortin-4 receptor (MC4R) pathway responsible for controlling energy balance, hunger, and satiety results in severe hyperphagia manifesting in food-seeking behaviors that drive the development of obesity early in childhood. These behaviors have negative impacts on many areas of the lives of patients with BBS and their families/caregivers, including sleep, mood, school/work, and social/family relationships. Additionally, many patients feel stigmatized due to their hyperphagia-associated food-seeking behaviors and the resulting obesity, which exacerbates the impacts of hyperphagia on quality of life. Early identification and management of hyperphagia in patients with BBS is key: mitigating food-seeking and weight gain can improve quality of life and reduce the risk of metabolic and cardiovascular diseases that is increased in patients with BBS. Until recently, the only treatment strategies available were lifestyle and diet modifications. However, targeted treatment with the novel MC4R agonist setmelanotide now offers an effective management option to reduce hyperphagia and weight in patients with BBS, improving overall health and quality of life.

The mutation S315T of the catalase-peroxidase (CP) protein KatG of Mycobacterium tuberculosis is the most common mutation that confers resistance to the prodrug isoniazid. Here, we reconstruct its evolutionary history in 145 whole-genome sequences of M. tuberculosis from Russian hospitals, inferring 11 independent appearances of this mutation and 5 reversion events, with an estimated reversion rate 1,500 times higher than the rate of preserved nonsynonymous or intragenic mutations. This suggests that, contrary to the commonly held view, the mutation KatG(S315T) results in a fitness cost, possibly because of reduced tolerance to oxidative stress. Consistent with this interpretation, the mutant enzyme presents reduced CP activities. Applying the torsional network model (TNM), we found that the mutant protein shows more restricted thermal dynamics, although its functional site moves quite similarly to the wild type. Of the four internal clones where KatG(S315T) arose, two present high reproductive rates and secondary mutations at the 5'-UTR region of the gene encoding superoxide dismutase A (sodA), while the other two present significantly lower reproductive rates and lack mutations at genes related with tolerance to oxidative stress. Our results suggest that the resistance mutation KatG(S315T) incurs a fitness cost, which may be alleviated through compensatory mutations at the gene sodA or other genes that respond to oxidative stress, such as the previously known gene ahpC. This suggests that isoniazid treatment could be complemented with drugs that produce oxidative stress in order to hinder the propagation of resistant strains devoid of compensatory mutations.

Cilia are essential organelles, and variants in genes governing ciliary function result in ciliopathic diseases. The Ciliogenesis and PLANar polarity Effectors (CPLANE) protein complex is essential for ciliogenesis, and all but one subunit of the CPLANE complex have been implicated in human ciliopathy. Here, we identify three families in which variants in the remaining CPLANE subunit CPLANE2/RSG1 also cause ciliopathy. These patients display cleft palate, tongue lobulations and polydactyly, phenotypes characteristic of Oral-Facial-Digital Syndrome. We further show that these alleles disrupt two vital steps of ciliogenesis, basal body docking and recruitment of intraflagellar transport proteins. Moreover, APMS reveals that Rsg1 binds CPLANE and the transition zone protein Fam92 in a GTP-dependent manner. Finally, we show that CPLANE is generally required for normal transition zone architecture. Our work demonstrates that CPLANE2/RSG1 is a causative gene for human ciliopathy and also sheds new light on the mechanisms of ciliary transition zone assembly.

BRAF and MEK inhibitors (BRAF/MEKi) have radically changed the treatment landscape of advanced BRAF mutation-positive tumours. However, limited efficacy and emergence of drug resistance are major barriers for successful treatments. Here, by using relevant preclinical models, we find that Connexin43 (Cx43), a protein that plays a role in cell-to-cell communication, enhances the effectiveness of BRAF/MEKi by recruiting DNA repair complexes to lamin-associated domains and promoting persistent DNA damage and cellular senescence. The nuclear compartmentalization promoted by Cx43 contributes to genome instability and synthetic lethality caused by excessive DNA damage, which could provide a therapeutic approach for these tumours to overcome drug resistance. Based on these findings, we designed a drug combination using small extracellular vesicles (sEVs) to deliver the full-Cx43 in combination with the BRAF/MEKi. This study reveals Cx43 as a regulator of DNA repair and BRAF/MEKi response, highlighting the therapeutic potential that this approach could eventually have in the clinic to overcome the limitations of current therapies and improve treatment outcomes for patients with advanced BRAF mutant tumours.

The spread of SARS-CoV-2 has been influenced by multiple factors, from the inherent transmission capabilities of the different variants to the control measurements put in place. Understanding how new variants enter a country is essential for managing future outbreaks. This study investigates how three major variants-Alpha, Delta, and Omicron (BA.1)-entered Spain and how different restrictions potentially affected their introduction.

Human virome studies are gaining attention as viruses are increasingly acknowledged as key modulators of microbial communities and human health. However, viral metagenomics presents distinct challenges, including the low abundance and diversity of viruses in biological samples, the lack of universal marker genes, and protocol-induced biases. Although various virome protocols have been benchmarked using viral particles or nucleic acids from mock communities, these often fail to replicate the complexity and heterogeneity of natural viromes. In this study, we systematically evaluated protocol modifications for the metagenomic analysis of human fecal samples, testing alternatives for viral enrichment, nucleic acid extraction, genome amplification, and library preparation. We assessed the impact of each modification on key inferences, including taxonomic and functional assignment, contig quality, viral diversity, and genome structure. Our results highlight important trade-offs between viral genome recovery and contamination removal, underscoring how methodological choices can shape virome composition. Based on our findings, we propose an optimized protocol that enhances the recovery of viral DNA and RNA genomes while minimizing contamination from non-viral sequences, providing a robust framework for future gut virome studies.

Single-cell genomics enables studying tissues and organisms at the highest resolution. However, since a cell contains a small amount of DNA, single-cell DNA sequencing (scDNA-seq) typically requires single-cell whole-genome amplification (scWGA). Unfortunately, scWGA methods introduce technical biases that complicate the interpretation of scDNA-seq data. We compared six scWGA methods, three MDA (multiple displacement amplification; GenomiPhi, REPLI-g, and TruePrime) and three non-MDA (Ampli1, MALBAC, and PicoPLEX), on 206 tumoral and 24 healthy human cells. scWGA methods performed differently depending on the parameter of interest. REPLI-g minimized regional amplification bias, while non-MDA methods showed a more uniform and reproducible amplification. Ampli1 exhibited the lowest allelic imbalance and dropout, the most accurate insertion or deletion (indel) and copy-number detection, and a low polymerase error rate. However, REPLI-g yielded higher DNA quantities, longer amplicons, and greater genome coverage. We offer a comprehensive guide for selecting a scWGA approach, outlining trade-offs that influence the interpretation of scDNA-seq data.

Substitution models of protein evolution describe the patterns of amino acid substitutions over evolutionary time and are fundamental for probabilistic methods of phylogenetic inference. At the protein level, a variety of substitution models are available, but only empirical substitution models are well established in phylogenetics due to their mathematical simplicity. Despite their importance, a database compiling the large number of currently available empirical substitution models of protein evolution is lacking, although such a resource could facilitate access, assessment, and subsequent implementation of these models into phylogenetic frameworks. Besides, little is known about formal comparisons between the current set of empirical substitution models. We present EModelDB, a database of empirical substitution models of protein evolution required for probabilistic protein phylogenetics that includes the corresponding exchangeability matrices, model classification, and model-specific biological information. The database is integrated into a graphical user interface, written in Python and SQL, that facilitates its usability. We also compared common empirical substitution models in terms of the distance between their relative rates of amino acid substitution and amino frequencies at equilibrium. We found that substitution models derived from proteins related in nature tend to cluster together, reflecting similar evolutionary patterns. Indeed, we evaluated the empirical substitution models in terms of the folding stability of the derived modeled proteins and found that they generally produce less stable proteins compared to real proteins, suggesting that substitution models with additional evolutionary constraints can be preferred for studying protein evolution accounting for folding stability. Database URL: https://github.com/Paula-Iglesias-Rivas/EModelDB.

Cancer cell lines are valuable models for studying tumor biology, yet their genomic evolution during culture can compromise experimental reproducibility. We conducted a detailed genomic analysis of the triple-negative breast cancer cell line MDA-MB-231-luc-GFP, examining sublines obtained from different sources, at various time points, and across distinct passages. We introduce the concept of intraline heterogeneity (ILH) to highlight the genomic variability observed among these sublines. Our analyses revealed extensive genomic diversity, including differences in single-nucleotide variants (SNVs) and copy number alterations (CNAs). In particular, CNAs exhibited remarkable heterogeneity, with pronounced chromosomal gains and losses between sublines, underscoring the impact of genomic instability on ILH. These findings suggest that ILH may influence experimental outcomes, emphasizing the importance of considering passage-specific genomic characterization to ensure consistency and reliability in cancer research.

Epilepsy is a common and severe neurological disease characterized by spontaneous and recurrent seizures. Although anti-seizure treatments are effective for most patients, approximately 30% remain pharmacoresistant. Moreover, uncontrolled seizures are associated with increased health risks and shortened life expectancy in individuals with refractory epilepsy. Preclinical studies play a crucial role in drug discovery, and the zebrafish (Danio rerio) have been successfully employed for this purpose. In this study, we utilized the zebrafish PTZ-induced seizure model to evaluate the effects of two peptides on seizure responses: Tripeptide (p-BTX-I) and the CX2 (a Cx43derivated peptide). Zebrafish larvae at 6 days post-fertilization were pre-treated with these peptides at various concentrations, depending on their experimental groups, 24h prior to seizure induction. We assessed seizure frequency, quantified swimming activity, measured transcript levels of genes related to inflammation and apoptosis (il1b, tnfa, cox1, cox2a, il6, casp3a, casp9, baxa, bcl2a, and c-fos), and analyzed the biodistribution of both peptides. Our results indicate that the Tripeptide exhibited anti-inflammatory and anti-apoptotic effects, particularly through reducing the expression of il1b and casp9. CX2 pre-treatment significantly downregulated inflammatory markers (il1b, il6, tnfa, and cox1). Biodistribution analysis confirmed that the CX2 peptide reached the zebrafish brain, suggesting a direct role in modulating seizure-related pathways. Our findings demonstrate that Tripeptide and CX2 peptides can modulate gene expression and mitigate molecular response associated with epileptic seizures in the zebrafish brain. These peptides thus represent promising candidates for future research aimed at developing novel anti-epileptic therapies. However, additional studies are required to evaluate their long-term efficacy, elucidate underlying mechanisms of action, and explore potential translational applications.

Four European Reference Networks (ERN-EYE, ERKNet, Endo-ERN, ERN-ITHACA) have teamed up to establish a consensus statement and recommendations for Bardet-Biedl syndrome (BBS). BBS is an autosomal recessive ciliopathy with at least 26 genes identified to date. The clinical manifestations are pleiotropic, can be observed in utero and will progress with age. Genetic testing has progressively improved in the last years prompting for a revision of the diagnostic criteria taking into account clinical Primary and Secondary features, as well as positive or negative molecular diagnosis. This consensus statement also emphasizes on initial diagnosis, monitoring and lifelong follow-up, and symptomatic care that can be provided to patients and family members according to the involved care professionals. For paediatricians, developmental anomalies can be at the forefront for diagnosis (such as polydactyly) but can require specific care, such as for associated neuro developmental disorders. For ophthalmology, the early onset retinal degeneration requires ad hoc functional and imaging technologies and specific care for severe visual impairment. For endocrinology, among other manifestations, early onset obesity and its complications has benefited from better evaluation of eating behaviour problems, improved lifestyle programs, and from novel pharmacological therapies. Kidney and urinary track involvements warrants lifespan attention, as chronic kidney failure can occur and early management might improve outcome. This consensus recommends revised diagnostic criteria for BBS that will ensure certainty of diagnosis, giving robust grounds for genetic counselling as well as in the perspective of future trials for innovative therapies.

The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Different factors influence the spread of SARS-CoV-2, from the inherent transmission capabilities of the different variants to the control measurements put in place. Here we studied the introduction of the Alpha, Delta, and Omicron-BA.1 variants of concern (VOCs) into Spain. For this, we collected genomic data from the GISAID database and combined it with connectivity data from different countries with Spain to perform a phylodynamic Bayesian analysis of the introductions. Our findings reveal that the introductions of these VOCs predominantly originated from France, especially in the case of Alpha. As travel restrictions were eased during the Delta and Omicron-BA.1 waves, the number of introductions from distinct countries increased, with the United Kingdom and Germany becoming significant sources of the virus. The largest number of introductions detected corresponded to the Delta wave, which was associated with fewer restrictions and the summer period, when Spain receives a considerable number of tourists. This research underscores the importance of monitoring international travel patterns and implementing targeted public health measures to manage the spread of SARS-CoV-2.

Addressing disease remission and treatment adherence in inflammatory bowel diseases (IBDs), such as Crohn's disease, poses significant challenges due to underlying oxidative and inflammatory processes. Nanotechnology emerges as a promising avenue for enhancing therapeutic outcomes in IBD by optimizing drug bioactivity, reducing toxicity, and extending circulation time. Gold nanoparticles, known for their resistance to gastrointestinal pH and possessing antioxidant and anti-inflammatory properties, offer particular promise. They can be produced by green synthesis with seaweed (ES), itself associated with gastroprotective and anti-inflammatory activities. In a murine model of Crohn's disease induced with 8% acetic acid, pretreatment with dexamethasone (0.2 mL/30 g) or Au@ES (25 and 50 mg/kg) effectively mitigated inflammatory features. Notably, ES (50 mg/kg) and Au@ES (50 mg/kg) administration resulted in significant reductions in both macroscopic and microscopic inflammation scores compared to the disease control group. Furthermore, these treatments normalized inflammatory cytokine expression while safeguarding myenteric plexus glial cells. They also impeded neutrophil activation, leading to reduced myeloperoxidase activity and lipid peroxidation, coupled with increased glutathione levels. In conclusion, ES and Au@ES exhibit potent efficacy in counteracting inflammation and oxidation processes in an experimental Crohn's disease model, suggesting their potential as alternative therapeutic strategies for IBD.

Wastewater surveillance for SARS-CoV-2 provides early warnings of emerging variants of concerns and can be used to screen for novel cryptic linked-read mutations, which are co-occurring single nucleotide mutations that are rare, or entirely missing, in existing SARS-CoV-2 databases. While previous approaches have focused on specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and investigating their potential origin. We present Crykey, a tool for rapidly identifying rare linked-read mutations across the genome of SARS-CoV-2. We evaluated the utility of Crykey on over 3,000 wastewater and over 22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations in wastewater that represent potential circulating cryptic lineages, serving as a new computational tool for wastewater surveillance of SARS-CoV-2.

The selection among substitution models of molecular evolution is fundamental for obtaining accurate phylogenetic inferences. At the protein level, evolutionary analyses are traditionally based on empirical substitution models but these models make unrealistic assumptions and are being surpassed by structurally constrained substitution (SCS) models. The SCS models often consider site-dependent evolution, a process that provides realism but complicates their implementation into likelihood functions that are commonly used for substitution model selection.

Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme-substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

Alström syndrome (ALMS; #203800) is an ultrarare monogenic recessive disease. This syndrome is associated with variants in the gene, which encodes a centrosome-associated protein involved in the regulation of several ciliary and extraciliary processes, such as centrosome cohesion, apoptosis, cell cycle control and receptor trafficking. The type of variant associated with ALMS is mostly complete loss-of-function variants (97%) and they are mainly located in exons 8, 10 and 16 of the gene. Other studies in the literature have tried to establish a genotype-phenotype correlation in this syndrome with limited success. The difficulty in recruiting a large cohort in rare diseases is the main barrier to conducting this type of study.

Our study provides insights into the evolution of the coronavirus disease 2019 (COVID-19) pandemic in Malta, a highly connected and understudied country. We combined epidemiological and phylodynamic analyses to analyze trends in the number of new cases, deaths, tests, positivity rates, and evolutionary and dispersal patterns from August 2020 to January 2022. Our reconstructions inferred 173 independent severe acute respiratory syndrome coronavirus 2 introductions into Malta from various global regions. Our study demonstrates that characterizing epidemiological trends coupled with phylodynamic modeling can inform the implementation of public health interventions to help control COVID-19 transmission in the community.

Alström syndrome (ALMS) is a rare autosomal recessive disease that is associated with mutations in ALMS1 gene. The main clinical manifestations of ALMS are retinal dystrophy, obesity, type 2 diabetes mellitus, dilated cardiomyopathy and multi-organ fibrosis, characteristic in kidneys and liver. Depletion of the protein encoded by ALMS1 has been associated with the alteration of different processes regulated via the primary cilium, such as the NOTCH or TGF-β signalling pathways. However, the cellular impact of these deregulated pathways in the absence of ALMS1 remains unknown.

We present Crykey, a computational tool for rapidly identifying cryptic mutations of SARS-CoV-2. Specifically, we identify co-occurring single nucleotide mutations on the same sequencing read, called linked-read mutations, that are rare or entirely missing in existing databases, and have the potential to represent novel cryptic lineages found in wastewater. While previous approaches exist for identifying cryptic linked-read mutations from specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and for tens of thousands of samples and with increased scrutiny, given their potential to represent either artifacts or hidden SARS-CoV-2 lineages. Crykey fills this gap by identifying rare linked-read mutations that pass stringent computational filters to limit the potential for artifacts. We evaluate the utility of Crykey on >3,000 wastewater and >22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over a three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations representing potential cryptic lineages in wastewater.

Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes.

Pulmonary arterial hypertension (PAH) is an infrequent disorder characterized by high blood pressure in the pulmonary arteries. It may lead to premature death or the requirement for lung and/or heart transplantation. Genetics plays an important and increasing role in the diagnosis of PAH. Here, we report seven additional patients with variants in and a review of sixty previously described patients in the literature. Patients described in this study suffered with additional conditions including large septal defects, as described by other groups. Collectively, sixty-seven PAH patients have been reported so far with variants in , including missense and loss-of-function (LoF) variants. The majority of the loss-of-function variants found in were detected in the last exon of the gene. Meanwhile, most missense variants were located within exon one, suggesting a probable tolerated change at the amino terminal part of the protein. In addition, we reported two idiopathic PAH patients presenting with the same variant previously detected in five patients by other studies, suggesting a possible hot spot. Research conducted on PAH associated with congenital heart disease (CHD) indicated that variants in might be particularly prevalent in this subgroup, as two out of our seven additional patients presented with CHD. Further research is still necessary to clarify the precise association between the biological pathway of and the development of PAH.

Cell lineages accumulate somatic mutations during organismal development, potentially leading to pathological states. The rate of somatic evolution within a cell population can vary due to multiple factors, including selection, a change in the mutation rate, or differences in the microenvironment. Here, we developed a statistical test called the Poisson Tree (PT) test to detect varying evolutionary rates among cell lineages, leveraging the phylogenetic signal of single-cell DNA sequencing (scDNA-seq) data. We applied the PT test to 24 healthy and cancer samples, rejecting a constant evolutionary rate in 11 out of 15 cancer and five out of nine healthy scDNA-seq datasets. In six cancer datasets, we identified subclonal mutations in known driver genes that could explain the rate accelerations of particular cancer lineages. Our findings demonstrate the efficacy of scDNA-seq for studying somatic evolution and suggest that cell lineages often evolve at different rates within cancer and healthy tissues.

Reduced expression and/or activity of Kv1.5 channels (encoded by ) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional variants in the PAH population analyzed was around 1%. The data indicate that some variants found in patients with PAH have critical consequences for channel function, supporting the idea that pathogenic variants may be a causative or contributing factor for PAH.

Bacterial and fungal infections are a challenging global problem due to the reported increasing resistance of pathogenic microorganisms to conventional antimicrobials. Nanomaterials are a promising strategy to fight infections caused by multidrug-resistant microbes. In this work, gold (Au@UP) and silver (Ag@UP) nanoparticles were produced for the first time by green synthesis using an aqueous extract of the invasive macroalgae (UP). The nanoparticles were characterized by a wide range of physicochemical techniques. Au@UP and Ag@UP demonstrated to be spherical and crystalline with an average size of 6.8 ± 1.0 nm and 14.1 ± 2.8 nm, respectively. Carbohydrates and proteins of the UP extract may participate in the synthesis and capping of the nanoparticles. The UP extract, Ag@UP, and Au@UP were assessed for their antimicrobial activity against , , and Ag@UP showed the highest antimicrobial activity with very low MIC and MBC values for all the tested bacteria, and Au@UP demonstrated to be very effective against biofilm-producing bacteria. The antifungal properties of both Ag@UP and Au@UP were remarkable, inhibiting hyphae formation. This study points towards a very promising biomedical exploitation of this invasive brown algae.

Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 10 and 10 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.

Antimicrobial resistance is an ever-growing global concern to public health with no clear or immediate solution. Silver nanoparticles (AgNPs) have long been proposed as efficient agents to fight the growing number of antibiotic-resistant strains. However, the synthesis of these particles is often linked to high costs and the use of toxic, hazardous chemicals, with environmental and health impact. In this study, we successfully produced AgNPs by green synthesis with the aid of the extract of two brown algae-Cystoseira baccata (CB) and Cystoseira tamariscifolia (CT)-and characterized their physico-chemical properties. The NPs produced in both cases (Ag@CB and Ag@CT) present similar sizes, with mean diameters of around 22 nm. The antioxidant activity of the extracts and the NPs was evaluated, with the extracts showing important antioxidant activity. The bacteriostatic and bactericidal properties of both Ag@CB and Ag@CT were tested and compared with gold NPs produced in the same algae extracts as previously reported. AgNPs demonstrated the strongest bacteriostatic and bactericidal properties, at concentrations as low as 2.16 µg/mL against Pseudomonas aeruginosa and Escherichia coli. Finally, the capacity of these samples to prevent the formation of biofilms characteristic of infections with a poorer outcome was assessed, obtaining similar results. This work points towards an alternative for the treatment of bacterial infections, even biofilm-inducing, with the possibility of minimizing the risk of drug resistance, albeit the necessary caution implied using metallic NPs.

Risk stratification in pulmonary arterial hypertension (PAH) is essential to provide more aggressive treatment for patients at higher risk. Nevertheless, recently introduced simplified prognostic tools neglect the genetic background. Additionally, pulmonary veno-oclusive disease (PVOD) has never been considered in risk assessment strategies.

Probabilistic phylogenetic tree reconstruction is traditionally performed under a best-fitting substitution model of molecular evolution previously selected according to diverse statistical criteria. Interestingly, some recent studies proposed that this procedure is unnecessary for phylogenetic tree reconstruction leading to a debate in the field. In contrast to DNA sequences, phylogenetic tree reconstruction from protein sequences is traditionally based on empirical exchangeability matrices that can differ among taxonomic groups and protein families. Considering this aspect, here we investigated the influence of selecting a substitution model of protein evolution on phylogenetic tree reconstruction by the analyses of real and simulated data. We found that phylogenetic tree reconstructions based on a selected best-fitting substitution model of protein evolution are the most accurate, in terms of topology and branch lengths, compared with those derived from substitution models with amino acid replacement matrices far from the selected best-fitting model, especially when the data has large genetic diversity. Indeed, we found that substitution models with similar amino acid replacement matrices produce similar reconstructed phylogenetic trees, suggesting the use of substitution models as similar as possible to a selected best-fitting model when the latter cannot be used. Therefore, we recommend the use of the traditional protocol of selection among substitution models of evolution for protein phylogenetic tree reconstruction.

Type IB topoisomerases relax the torsional stress associated with DNA metabolism in the nucleus and mitochondria and constitute important molecular targets of anticancer drugs. Vertebrates stand out among eukaryotes by having two Type IB topoisomerases acting specifically in the nucleus (TOP1) and mitochondria (TOP1MT). Despite their major importance, the origin and evolution of these paralogues remain unknown. Here, we examine the molecular evolutionary processes acting on both TOP1 and TOP1MT in Chordata, taking advantage of the increasing number of available genome sequences. We found that both TOP1 and TOP1MT evolved under strong purifying selection, as expected considering their essential biological functions. Critical active sites, including those associated with resistance to anticancer agents, were found particularly conserved. However, TOP1MT presented a higher rate of molecular evolution than TOP1, possibly related with its specialized activity on the mitochondrial genome and a less critical role in cells. We could place the duplication event that originated the TOP1 and TOP1MT paralogues early in the radiation of vertebrates, most likely associated with the first round of vertebrate tetraploidization (1R). Moreover, our data suggest that cyclostomes present a specialized mitochondrial Type IB topoisomerase. Interestingly, we identified two missense mutations replacing amino acids in the Linker region of TOP1MT in Neanderthals, which appears as a rare event when comparing the genome of both species. In conclusion, TOP1 and TOP1MT differ in their rates of evolution, and their evolutionary histories allowed us to better understand the evolution of chordates.

Genetic recombination is a common evolutionary mechanism that produces molecular diversity. However, its consequences on protein folding stability have not attracted the same attention as in the case of point mutations. Here, we studied the effects of homologous recombination on the computationally predicted protein folding stability for several protein families, finding less detrimental effects than we previously expected. Although recombination can affect multiple protein sites, we found that the fraction of recombined proteins that are eliminated by negative selection because of insufficient stability is not significantly larger than the corresponding fraction of proteins produced by mutation events. Indeed, although recombination disrupts epistatic interactions, the mean stability of recombinant proteins is not lower than that of their parents. On the other hand, the difference of stability between recombined proteins is amplified with respect to the parents, promoting phenotypic diversity. As a result, at least one third of recombined proteins present stability between those of their parents, and a substantial fraction have higher or lower stability than those of both parents. As expected, we found that parents with similar sequences tend to produce recombined proteins with stability close to that of the parents. Finally, the simulation of protein evolution along the ancestral recombination graph with empirical substitution models commonly used in phylogenetics, which ignore constraints on protein folding stability, showed that recombination favors the decrease of folding stability, supporting the convenience of adopting structurally constrained models when possible for inferences of protein evolutionary histories with recombination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) produced diverse molecular variants during its recent expansion in humans that caused different transmissibility and severity of the associated disease as well as resistance to monoclonal antibodies and polyclonal sera, among other treatments. In order to understand the causes and consequences of the observed SARS-CoV-2 molecular diversity, a variety of recent studies investigated the molecular evolution of this virus during its expansion in humans. In general, this virus evolves with a moderate rate of evolution, in the order of 10-10 substitutions per site and per year, which presents continuous fluctuations over time. Despite its origin being frequently associated with recombination events between related coronaviruses, little evidence of recombination was detected, and it was mostly located in the spike coding region. Molecular adaptation is heterogeneous among SARS-CoV-2 genes. Although most of the genes evolved under purifying selection, several genes showed genetic signatures of diversifying selection, including a number of positively selected sites that affect proteins relevant for the virus replication. Here, we review current knowledge about the molecular evolution of SARS-CoV-2 in humans, including the emergence and establishment of variants of concern. We also clarify relationships between the nomenclatures of SARS-CoV-2 lineages. We conclude that the molecular evolution of this virus should be monitored over time for predicting relevant phenotypic consequences and designing future efficient treatments.

Bardet-Biedl syndrome (BBS) is a rare genetic disease of the group of ciliopathies, a group of pathologies characterized mainly by defects in the structure and/or function of primary cilia. The main features of this ciliopathy are retinal dystrophy, obesity, polydactyly, urogenital and renal abnormalities, and cognitive impairment, commonly accompanied by various secondary features, making clear the extensive clinical heterogeneity associated with this syndrome, which, together with the frequent overlapping phenotype with other ciliopathies, greatly complicates its diagnosis. Patients are mainly detected by their pediatrician at quite early ages, usually between 2 and 6years. The pediatrician, given the main symptoms they present, usually refers patients to a specialist. Personalized medicine brought diagnosis closer to many patients who lacked it. It usually presents an autosomal recessive mode of inheritance, but in recent years several authors have proposed more complex inheritance models to explain the frequent inter- and intra-familial clinical variability. The main molecular techniques used for diagnosis are gene panels, the clinical exome and, in certain cases, the patient's complete genome. Although numerous studies have contributed to defining the role of the different BBS genes and designing various strategies for the molecular diagnosis of BBS, as well as delving into the functions performed by these proteins, these advances have not been sufficient to develop a complete treatment for this syndrome. and to be able to offer patients some therapeutic options.

Risk stratification in pulmonary arterial hypertension (PAH) is essential to provide more aggressive treatment for patients at higher risk. Nevertheless, recently introduced simplified prognostic tools neglect the genetic background. Additionally, pulmonary veno-oclusive disease (PVOD) has never been considered in risk assessment strategies.

Genetic recombination is a common evolutionary mechanism that produces molecular diversity. However, its consequences on protein folding stability have not attracted the same attention as in the case of point mutations. Here, we studied the effects of homologous recombination on the computationally predicted protein folding stability for several protein families, finding less detrimental effects than we previously expected. Although recombination can affect multiple protein sites, we found that the fraction of recombined proteins that are eliminated by negative selection because of insufficient stability is not significantly larger than the corresponding fraction of proteins produced by mutation events. Indeed, although recombination disrupts epistatic interactions, the mean stability of recombinant proteins is not lower than that of their parents. On the other hand, the difference of stability between recombined proteins is amplified with respect to the parents, promoting phenotypic diversity. As a result, at least one third of recombined proteins present stability between those of their parents, and a substantial fraction have higher or lower stability than those of both parents. As expected, we found that parents with similar sequences tend to produce recombined proteins with stability close to that of the parents. Finally, the simulation of protein evolution along the ancestral recombination graph with empirical substitution models commonly used in phylogenetics, which ignore constraints on protein folding stability, showed that recombination favors the decrease of folding stability, supporting the convenience of adopting structurally constrained models when possible for inferences of protein evolutionary histories with recombination.

Despite the increased recognition of (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. To compile and functionally characterize all variants reported to date and undertake a comprehensive genotype-phenotype analysis. We assembled a multicenter cohort of 137 patients harboring monoallelic variants and assessed the pathogenicity of missense variation ( = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with (Bone Morphogenetic Protein Receptor type 2) causal variants ( = 162) or no identified variants in PAH-associated genes ( = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Functional assessment of missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects ( = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation ( = 0.005) and increased incidence of interstitial lung disease ( = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group ( = 0.022), although age had a significant effect in the hazard model ( = 0.0461). Carriers of variants were diagnosed at a younger age ( < 0.001) and had worse baseline lung function (FEV, FVC) ( = 0.009) than the and no identified causal variant groups. We demonstrated that syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

Cancer still poses a global threat, since a lot of tumors remain untreatable despite all the available chemotherapeutic drugs, whose side effects, it must also be noted, still raise concerns. The antitumoral properties of marine seaweeds make them a potential source of new, less toxic, and more active antitumoral agents. Furthermore, these natural extracts can be combined with nanotechnology to increase their efficacy and improve targeting. In this work, a (CT) aqueous extract was employed for the green synthesis of gold nanoparticles (Au@CT). The complete characterization of Au@CT was performed by UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, Zeta potential, electron microscopy, X-ray powder diffraction (XRD), high-performance steric exclusion chromatography (HPSEC), and by the determination of their antioxidant capacity. The antiproliferative activity of Au@CT was then tested in hepatic (HEPG-2) and pancreatic (BxPC-3) cell lines. Their potential capacity as enhancers of gemcitabine, a drug frequently used to treat both types of tumors, was also tested. The activity of Au@CT was compared to the activity of the CT extract alone. A synergistic effect with gemcitabine was proven for HEPG-2. Our results showed that gold nanoparticles synthesized from seaweed extracts with antitumoral activity could be a good gemcitabine enhancer.

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.

We present SIEVE, a statistical method for the joint inference of somatic variants and cell phylogeny under the finite-sites assumption from single-cell DNA sequencing. SIEVE leverages raw read counts for all nucleotides and corrects the acquisition bias of branch lengths. In our simulations, SIEVE outperforms other methods in phylogenetic reconstruction and variant calling accuracy, especially in the inference of homozygous variants. Applying SIEVE to three datasets, one for triple-negative breast (TNBC), and two for colorectal cancer (CRC), we find that double mutant genotypes are rare in CRC but unexpectedly frequent in the TNBC samples.

Recurrence of tumor cells following local and systemic therapy is a significant hurdle in cancer. Most patients with metastatic colorectal cancer (mCRC) will relapse, despite resection of the metastatic lesions. A better understanding of the evolutionary history of recurrent lesions is required to identify the spatial and temporal patterns of metastatic progression and expose the genetic and evolutionary determinants of therapeutic resistance. With this goal in mind, here we leveraged a unique single-cell whole-genome sequencing dataset from recurrent hepatic lesions of an mCRC patient. Our phylogenetic analysis confirms that the treatment induced a severe demographic bottleneck in the liver metastasis but also that a previously diverged lineage survived this surgery, possibly after migration to a different site in the liver. This lineage evolved very slowly for two years under adjuvant drug therapy and diversified again in a very short period. We identified several non-silent mutations specific to this lineage and inferred a substantial contribution of chemotherapy to the overall, genome-wide mutational burden. All in all, our study suggests that mCRC subclones can migrate locally and evade resection, keep evolving despite rounds of chemotherapy, and re-expand explosively.

Due to their rarity and heterogeneity and despite the introduction of molecular features in the current WHO classification, clinical criteria such as those from the European Organization for Research and Treatment of Cancer (EORTC) and the Radiation Therapy Oncology Group (RTOG) are still being used to make treatment decisions in low-grade gliomas (LGG). Patients with diffuse low-grade glioma treated at our institution between 2002 and 2018 were analyzed, retrieving and assessing the degree of consistency between the EORTC and RTOG criteria, as well as the isocitrate dehydrogenase 1 and 2 (IDH) gene mutational status. Likewise, multivariate analyses were performed to ascertain the superiority of any of the factors over the others. One hundred and two patients were included. The degree of consistency between the RTOG and EORTC criteria was 71.6% (K = 0.426; p = 0.0001). Notably, 51.7% of those assigned to low risk by the EORTC were classified as high risk according to the RTOG classification. In multivariate analysis, only complete resection, age > 40 years, size and IDH mutation status were independently correlated with OS. When the RTOG and EORTC scores were entered into the model, only the EORTC model was independently associated with mortality. The degree of consistency between the EORT and RTOG criteria is low. Therefore, there is a need to integrate clinical-molecular scores to improve treatment decisions in LGG.

Advances in phylogenomics and high-throughput sequencing have allowed the reconstruction of deep phylogenetic relationships in the evolution of eukaryotes. Yet, the root of the eukaryotic tree of life remains elusive. The most popular hypothesis in textbooks and reviews is a root between Unikonta (Opisthokonta + Amoebozoa) and Bikonta (all other eukaryotes), which emerged from analyses of a single-gene fusion. Subsequent, highly cited studies based on concatenation of genes supported this hypothesis with some variations or proposed a root within Excavata. However, concatenation of genes does not consider phylogenetically-informative events like gene duplications and losses. A recent study using gene tree parsimony (GTP) suggested the root lies between Opisthokonta and all other eukaryotes, but only including 59 taxa and 20 genes. Here we use GTP with a duplication-loss model in a gene-rich and taxon-rich dataset (i.e., 2,786 gene families from two sets of 155 and 158 diverse eukaryotic lineages) to assess the root, and we iterate each analysis 100 times to quantify tree space uncertainty. We also contrasted our results and discarded alternative hypotheses from the literature using GTP and the likelihood-based method SpeciesRax. Our estimates suggest a root between Fungi or Opisthokonta and all other eukaryotes; but based on further analysis of genome size, we propose that the root between Opisthokonta and all other eukaryotes is the most likely.

The selection of the best-fitting substitution model of molecular evolution is a traditional step for phylogenetic inferences, including ancestral sequence reconstruction (ASR). However, a few recent studies suggested that applying this procedure does not affect the accuracy of phylogenetic tree reconstruction. Here, we revisited this debate topic by analyzing the influence of selection among substitution models of protein evolution, with focus on exchangeability matrices, on the accuracy of ASR using simulated and real data. We found that the selected best-fitting substitution model produces the most accurate ancestral sequences, especially if the data present large genetic diversity. Indeed, ancestral sequences reconstructed under substitution models with similar exchangeability matrices were similar, suggesting that if the selected best-fitting model cannot be used for the reconstruction, applying a model similar to the selected one is preferred. We conclude that selecting among substitution models of protein evolution is recommended for reconstructing accurate ancestral sequences.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBS-associated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS cases.

Single-nucleotide variants (SNVs) are the most common variations in the human genome. Recently developed methods for SNV detection from single-cell DNA sequencing data, such as SCIΦ and scVILP, leverage the evolutionary history of the cells to overcome the technical errors associated with single-cell sequencing protocols. Despite being accurate, these methods are not scalable to the extensive genomic breadth of single-cell whole-genome (scWGS) and whole-exome sequencing (scWES) data.

Primary cilia are non-motile organelles associated with the cell cycle, which can be found in most vertebrate cell types. Cilia formation occurs through a process called ciliogenesis, which involves several mechanisms including planar cell polarity (PCP) and the Hedgehog (Hh) signaling pathway. Some gene complexes, such as BBSome or CPLANE (ciliogenesis and planar polarity effector), have been linked to ciliogenesis. CPLANE complex is composed of , and , which bind to and for cilia formation. Defects in these genes have been linked to a malfunction of intraflagellar transport and defects in the planar cell polarity, as well as defective activation of the Hedgehog signalling pathway. These faults lead to defective cilium formation, resulting in ciliopathies, including orofacial-digital syndrome (OFDS) and Bardet-Biedl syndrome (BBS). Considering the close relationship, between the CPLANE complex and cilium formation, it can be expected that defects in the genes that encode subunits of the CPLANE complex may be related to other ciliopathies.

The integral utilization of sustainable resources with versatile, efficient and cleaner processes is encouraged. Hydrothermal treatment with subcritical water is a chemical free, tunable and rapid technology providing enhanced yield compared to conventional extraction and was explored for the benign by design extraction and depolymerization of carrageenan from Chondrus crispus. Up to 90% of the seaweed was solubilized operating under nonisothermal regime during heating up to 200 °C and 75.5% crude carrageenan yield was attained at 140 °C. Crude carrageenan could not be precipitated by ethanol from the extracts produced at 180 °C and higher temperatures, but ultrafiltration (100 kDa) of the extract obtained at 160 °C provided comparable recovery yields and similar rheological features to those of the ethanol precipitated product. Operation at 140 °C was preferred based on the higher recovery yield of the biopolymer and the whole extract was suitable for the green synthesis of polycrystalline decahedral quasi-spherical gold nanoparticles with a mean size distribution of 8.4 nm and Z potential value of -40.2 mV. Alternatively, the crude carrageen fraction was used for the formulation of printable biopolymer based gels with suitable mechanical properties, including a relevant gel strength enhancement (about 10-fold) when compared with conventional procedures.

TOPIIA topoisomerases are required for the regulation of DNA topology by DNA cleavage and re-ligation and are important targets of antibiotic and anticancer agents. Humans possess two TOPIIA paralogue genes (TOP2A and TOP2B) with high sequence and structural similarity but distinct cellular functions. Despite their functional and clinical relevance, the evolutionary history of TOPIIA is still poorly understood. Here we show that TOPIIA is highly conserved in Metazoa. We also found that TOPIIA paralogues from jawed and jawless vertebrates had different origins related with tetraploidization events. After duplication, TOP2B evolved under a stronger purifying selection than TOP2A, perhaps promoted by the more specialized role of TOP2B in postmitotic cells. We also detected genetic signatures of positive selection in the highly variable C-terminal domain (CTD), possibly associated with adaptation to cellular interactions. By comparing TOPIIA from modern and archaic humans, we found two amino acid substitutions in the TOP2A CTD, suggesting that TOP2A may have contributed to the evolution of present-day humans, as proposed for other cell cycle-related genes. Finally, we identified six residues conferring resistance to chemotherapy differing between TOP2A and TOP2B. These six residues could be targets for the development of TOP2A-specific inhibitors that would avoid the side effects caused by inhibiting TOP2B. Altogether, our findings clarify the origin, diversification and selection pressures governing the evolution of animal TOPIIA.

Human mitochondria can be genetically distinct within the same individual, a phenomenon known as heteroplasmy. In cancer, this phenomenon seems exacerbated, and most mitochondrial mutations seem to be heteroplasmic. How this genetic variation is arranged within and among normal and tumor cells is not well understood. To address this question, here we sequenced single-cell mitochondrial genomes from multiple normal and tumoral locations in four colorectal cancer patients. Our results suggest that single cells, both normal and tumoral, can carry various mitochondrial haplotypes. Remarkably, this intra-cell heteroplasmy can arise before tumor development and be maintained afterward in specific tumoral cell subpopulations. At least in the colorectal patients studied here, the somatic mutations in the single-cells do not seem to have a prominent role in tumorigenesis.

Long-Term Non-Progressors (LTNPs) are untreated Human Immunodeficiency virus type 1 (HIV-1) infected individuals able to control disease progression for prolonged periods. However, the LTNPs status is temporary, as viral load increases followed by decreases in CD4 + T-cell counts. Control of HIV-1 infection in LTNPs viremic controllers, have been associated with effective immunodominant HIV-1 Gag-CD8 + T-cell responses restricted by protective HLA-B alleles. Individuals carrying HLA-B*14:02 control HIV-1 infection is related to an immunodominant Env-CD8 + T-cell response. Limited data are available on the contribution of HLA-B*14:02 CD8 + T -cells in LTNPs.

The last glacial period (LGP) promoted a loss of genetic diversity in Paleolithic populations of modern humans from diverse regions of the world by range contractions and habitat fragmentation. However, this period also provided some currently submersed lands, such as the Sunda shelf in Southeast Asia (SEA), that could have favored the expansion of our species. Concerning the latter, still little is known about the influence of the lowering sea level on the genetic diversity of current SEA populations. Here, we applied approximate Bayesian computation, based on extensive spatially explicit computer simulations, to evaluate the fitting of mtDNA data from diverse SEA populations with alternative evolutionary scenarios that consider and ignore the LGP and migration through long-distance dispersal (LDD). We found that both the LGP and migration through LDD should be taken into consideration to explain the currently observed genetic diversity in these populations and supported a rapid expansion of first populations throughout SEA. We also found that temporarily available lands caused by the low sea level of the LGP provided additional resources and migration corridors that favored genetic diversity. We conclude that migration through LDD and temporarily available lands during the LGP should be considered to properly understand and model the first expansions of modern humans.

This study reports on the green and cost-efficient synthesis of gold nanoparticles from three different red algae extracts. The nanoparticles synthesized were fully characterized by UV-Vis spectroscopy, HRTEM, and Z-potential. Relevant components occurring in the extracts, such as polysaccharides or phenolic content, were assessed by analytical techniques such as spectrophotometric assays and liquid chromatography. Finally, the antioxidant, antitumoral, and anti-inflammatory potential of both the extracts and the gold nanoparticles synthesized were analyzed in order to determine a possible synergistic effect on the nanoparticles. The results obtained confirmed the obtainment of gold nanoparticles with significant potential as immunotherapeutic agents. The therapeutic potential of these nanoparticles could be higher than that of inert gold nanoparticles loaded with bioactive molecules since the former would allow for higher accumulation into the targeted tissue.

Clonally transmissible cancers are tumour lineages that are transmitted between individuals via the transfer of living cancer cells. In marine bivalves, leukaemia-like transmissible cancers, called hemic neoplasia (HN), have demonstrated the ability to infect individuals from different species. We performed whole-genome sequencing in eight warty venus clams that were diagnosed with HN, from two sampling points located more than 1000 nautical miles away in the Atlantic Ocean and the Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing analysis from neoplastic animals revealed the coexistence of haplotypes from two different clam species. Phylogenies estimated from mitochondrial and nuclear markers confirmed this leukaemia originated in striped venus clams and later transmitted to clams of the species warty venus, in which it survives as a contagious cancer. The analysis of mitochondrial and nuclear gene sequences supports all studied tumours belong to a single neoplastic lineage that spreads in the Seas of Southern Europe.

Intra-host evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in cases with persistent coronavirus disease 2019 (COVID-19). In this study, we describe a severely immunosuppressed individual with HIV-1/SARS-CoV-2 coinfection with a long-term course of SARS-CoV-2 infection. A 28-year-old man was diagnosed with HIV-1 infection (CD4+ count: 3 cells/µL nd 563000 HIV-1 RNA copies/mL) and simultaneous pneumonia, disseminated infection and SARS-CoV-2 infection. SARS-CoV-2 real-time reverse transcription polymerase chain reaction positivity from nasopharyngeal samples was prolonged for 15 weeks. SARS-CoV-2 was identified as variant Alpha (PANGO lineage B.1.1.7) with mutation S:E484K. Spike-specific T-cell response was similar to HIV-negative controls although enriched in IL-2, and showed disproportionately increased immunological exhaustion marker levels. Despite persistent SARS-CoV-2 infection, adaptive intra-host SARS-CoV-2 evolution, was not identified. Spike-specific T-cell response protected against a severe COVID-19 outcome and the increased immunological exhaustion marker levels might have favoured SARS-CoV-2 persistence.

We introduce CellPhy, a maximum likelihood framework for inferring phylogenetic trees from somatic single-cell single-nucleotide variants. CellPhy leverages a finite-site Markov genotype model with 16 diploid states and considers amplification error and allelic dropout. We implement CellPhy into RAxML-NG, a widely used phylogenetic inference package that provides statistical confidence measurements and scales well on large datasets with hundreds or thousands of cells. Comprehensive simulations suggest that CellPhy is more robust to single-cell genomics errors and outperforms state-of-the-art methods under realistic scenarios, both in accuracy and speed. CellPhy is freely available at https://github.com/amkozlov/cellphy .

Single-cell sequencing has gained popularity in recent years. Despite its numerous applications, single-cell DNA sequencing data is highly error-prone due to technical biases arising from uneven sequencing coverage, allelic dropout, and amplification error. With these artifacts, the identification of somatic genomic variants becomes a challenging task, and over the years, several methods have been developed explicitly for this type of data. Single-cell variant callers implement distinct strategies, make different use of the data, and typically result in many discordant calls when applied to real data. Here, we review current approaches for single-cell variant calling, emphasizing single nucleotide variants. We highlight their potential benefits and shortcomings to help users choose a suitable tool for their data at hand.

is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, cell adhesion and epithelial-mesenchymal transition (EMT). Nevertheless, the role of gene in fibrosis generation and other implicated processes such as cell migration or cell adhesion the TGF- β pathway has not been elucidated yet. Initially, we evaluated how depletion of affects different processes like apoptosis, cell cycle and mitochondrial activity in HeLa cells. Then, we performed proteomic profiling with TGF-β stimuli in HeLa -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers were also studied in hTERT-BJ-5ta -/-. Finally, we evaluated the SMAD3 and SMAD2 phosphorylation and cell migration capacity in both models. Depletion of generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between and . Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction in HeLa. showed an opposite pattern to what would be expected when activating the EMT in HeLa and BJ-5ta. Finally, in BJ-5ta model a reduced activation of SMAD3 but not SMAD2 were also observed. In HeLa model no alterations in the canonical TGF-β pathway were observed but both cell lines showed a reduction in migration capacity. has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of affects the signal transduction through the TGF-β and other processes like the cell migration and adhesion capacity.

Octocorals possess sclerites, small elements comprised of calcium carbonate (CaCO) that are important diagnostic characters in octocoral taxonomy. Among octocorals, sea pens comprise a unique order (Pennatulacea) that live in a wide range of depths. Habitat depth is considered to be important in the diversification of octocoral species, but a lack of information on sea pens has limited studies on their adaptation and evolution across depth. Here, we aimed to reveal trends of adaptation and evolution of sclerite shapes in sea pens with regards to habitat depth phylogenetic analyses and ancestral reconstruction analyses. Colony form of sea pens is suggested to have undergone convergent evolution and the loss of axis has occurred independently across the evolution of sea pens. Divergences of sea pen taxa and of sclerite forms are suggested to depend on habitat depths. In addition, their sclerite forms may be related to evolutionary history of the sclerite and the surrounding chemical environment as well as water temperature. Three-flanged sclerites may possess the tolerance towards the environment of the deep sea, while plate sclerites are suggested to be adapted towards shallower waters, and have evolved independently multiple times. The common ancestor form of sea pens was predicted to be deep-sea and similar to family Pseudumbellulidae in form, possessing sclerites intermediate in form to those of alcyonaceans and modern sea pens such as spindles, rods with spines, and three-flanged sclerites with serrated edges sclerites, as well as having an axis and bilateral traits.

The reconstruction of genetic material of ancestral organisms constitutes a powerful application of evolutionary biology. A fundamental step in this inference is the ancestral sequence reconstruction (ASR), which can be performed with diverse methodologies implemented in computer frameworks. However, most of these methodologies ignore evolutionary properties frequently observed in microbes, such as genetic recombination and complex selection processes, that can bias the traditional ASR. From a practical perspective, here I review methodologies for the reconstruction of ancestral DNA and protein sequences, with particular focus on microbes, and including biases, recommendations, and software implementations. I conclude that microbial ASR is a complex analysis that should be carefully performed and that there is a need for methods to infer more realistic ancestral microbial sequences.

A detailed understanding of how and when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission occurs is crucial for designing effective prevention measures. Other than contact tracing, genome sequencing provides information to help infer who infected whom. However, the effectiveness of the genomic approach in this context depends on both (high enough) mutation and (low enough) transmission rates. Today, the level of resolution that we can obtain when describing SARS-CoV-2 outbreaks using just genomic information alone remains unclear. In order to answer this question, we sequenced forty-nine SARS-CoV-2 patient samples from ten local clusters in NW Spain for which partial epidemiological information was available and inferred transmission history using genomic variants. Importantly, we obtained high-quality genomic data, sequencing each sample twice and using unique barcodes to exclude cross-sample contamination. Phylogenetic and cluster analyses showed that consensus genomes were generally sufficient to discriminate among independent transmission clusters. However, levels of intrahost variation were low, which prevented in most cases the unambiguous identification of direct transmission events. After filtering out recurrent variants across clusters, the genomic data were generally compatible with the epidemiological information but did not support specific transmission events over possible alternatives. We estimated the effective transmission bottleneck size to be one to two viral particles for sample pairs whose donor-recipient relationship was likely. Our analyses suggest that intrahost genomic variation in SARS-CoV-2 might be generally limited and that homoplasy and recurrent errors complicate identifying shared intrahost variants. Reliable reconstruction of direct SARS-CoV-2 transmission based solely on genomic data seems hindered by a slow mutation rate, potential convergent events, and technical artifacts. Detailed contact tracing seems essential in most cases to study SARS-CoV-2 transmission at high resolution.

The evolution of structural proteins is generally constrained by the folding stability. However, little is known about the particular capacity of viral proteins to accommodate mutations that can potentially affect the protein stability and, in general, the evolution of the protein stability over time. As an illustrative model case, here, we investigated the evolution of the stability of the human immunodeficiency virus (HIV-1) protease (PR), which is a common HIV-1 drug target, under diverse evolutionary scenarios that include (1) intra-host virus evolution in a cohort of seventy-five patients sampled over time, (2) intra-host virus evolution sampled before and after specific PR-based treatments, and (3) inter-host evolution considering extant and ancestral (reconstructed) PR sequences from diverse HIV-1 subtypes. We also investigated the specific influence of currently known HIV-1 PR resistance mutations on the PR folding stability. We found that the HIV-1 PR stability fluctuated over time within a constant and wide range in any studied evolutionary scenario, accommodating multiple mutations that partially affected the stability while maintaining activity. We did not identify relationships between change of PR stability and diverse clinical parameters such as viral load, CD4 T-cell counts, and a surrogate of time from infection. Counterintuitively, we predicted that nearly half of the studied HIV-1 PR resistance mutations do not significantly decrease stability, which, together with compensatory mutations, would allow the protein to adapt without requiring dramatic stability changes. We conclude that the HIV-1 PR presents a wide structural plasticity to acquire molecular adaptations without affecting the overall evolution of stability.

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect , and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity depending on their genotype. We determined that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied.