Sequence Evolution Research Articles

Linear motifs regulating protein secretion, sorting and autophagy in Leishmania parasites are diverged with respect to their host equivalents.

The pathogenic, tropical Leishmania flagellates belong to an early-branching eukaryotic lineage (Kinetoplastida) with several unique features. Unfortunately, they are poorly understood from a molecular biology perspective, making development of mechanistically novel and selective drugs difficult. Here, we explore three functionally critical targeting short linear motif systems as well as their receptors in depth, using a combination of structural modeling, evolutionary sequence divergence and deep learning. Secretory signal peptides, endoplasmic reticulum (ER) retention motifs (KDEL motifs), and autophagy signals (motifs interacting with ATG8 family members) are ancient and essential components of cellular life. Although expected to be conserved amongst the kinetoplastids, we observe that all three systems show a varying degree of divergence from their better studied equivalents in animals, plants, or fungi. We not only describe their behaviour, but also build models that allow the prediction of localization and potential functions for several uncharacterized Leishmania proteins. The unusually Ala/Val-rich secretory signal peptides, endoplasmic reticulum resident proteins ending in Asp-Leu-COOH and atypical ATG8-like proteins are all unique molecular features of kinetoplastid parasites. Several of their critical protein-protein interactions could serve as targets of selective antimicrobial agents against Leishmaniasis due to their systematic divergence from the host.

Positive selection and relaxed purifying selection contribute to rapid evolution of male-biased genes in a dioecious flowering plant.

Sex-biased genes offer insights into the evolution of sexual dimorphism. Sex-biased genes, especially those with male bias, show elevated evolutionary rates of protein sequences driven by positive selection and relaxed purifying selection in animals. Although rapid sequence evolution of sex-biased genes and evolutionary forces have been investigated in animals and brown algae, less is known about evolutionary forces in dioecious angiosperms. In this study, we separately compared the expression of sex-biased genes between female and male floral buds and between female and male flowers at anthesis in dioecious Trichosanthes pilosa (Cucurbitaceae). In floral buds, sex-biased gene expression was pervasive, and had significantly different roles in sexual dimorphism such as physiology. We observed higher rates of sequence evolution for male-biased genes in floral buds compared to female-biased and unbiased genes. Male-biased genes under positive selection were mainly associated with functions to abiotic stress and immune responses, suggesting that high evolutionary rates are driven by adaptive evolution. Additionally, relaxed purifying selection may contribute to accelerated evolution in male-biased genes generated by gene duplication. Our findings, for the first time in angiosperms, suggest evident rapid evolution of male-biased genes, advance our understanding of the patterns and forces driving the evolution of sexual dimorphism in dioecious plants.

New insights into the effect of pyrolysis temperature on the spectroscopy properties of dissolved organic matter in manure-based biochar.

Dissolved organic matter (DOM) derived from biochar takes a crucial role in transport and bioavailability toward contaminants; hence, it is undeniable that a thorough analysis of its properties is important. So far, the effect of pyrolysis temperature on the functional groups, components, and evolutionary sequence of manure-based biochar DOM has not been adequately investigated. Here, DOM was released from two typical livestock manures (cow and pig) at five pyrolysis temperatures (300 ~ 700°C), and it was explored in depth with the aid of moving window 2D correlation spectroscopy (MW-2D-COS) and heterogeneous 2D correlation spectroscopy (hetero-2D-COS). The results demonstrated that the concentration, aromaticity, and hydrophobicity of DOM were greater at high temperatures, and more DOM was liberated from cow manure-based biochar at identical temperature. Protein-like compounds dominated at high temperatures. The pyrolysis temperatures of final configuration transformation points of the fulvic acid-like component and the aromatic ring C=C in DOM were 400°C and 500°C, respectively. Moreover, Fourier transform infrared spectroscopy combined with two-dimensional correlation analysis indicated that the functional group evolution of DOM depends on the pyrolysis temperature and feedstock type. The study provides a new perspective on manure management and environmental applications of biochar.

Evolution of gas flows along the starburst to post-starburst to quiescent galaxy sequence

ABSTRACT We measure velocity offsets in the $\rm{Na {}\rm {\small I}}$ λλ5890, 5896 ($\rm{Na {}\rm {\small D}}$) interstellar medium absorption lines to track how neutral galactic winds change as their host galaxies evolve. Our sample of ∼80 000 SDSS spectra at 0.010 < z < 0.325 includes starburst, post-starburst, and quiescent galaxies, forming an evolutionary sequence of declining star formation rate (SFR). We detect bulk flows across this sequence, mostly at higher host stellar masses (log(M*/M⊙)) > 10). Along this sequence, the fraction of outflows decreases (76 ± 2 per cent to 65 ± 4 per cent to a 3σ upper limit of 34 per cent), and the mean velocity offset changes from outflowing to inflowing (−84.6 ± 5.9 to −71.6 ± 11.4 to $76.6\pm 2.3\, \rm km s^{-1}$). Even within the post-starburst sample, wind speed decreases with time elapsed since the starburst ended. These results reveal that outflows diminish as galaxies age. For post-starbursts, there is evidence for an AGN contribution, especially to the speediest outflows: (1) SFR declines faster in time than outflow velocity, a decoupling arguing against massive stellar feedback; (2) of the few outflows strong enough to escape the interstellar medium (9/105), three of the four hosts with measured emission lines are Seyfert galaxies. For discy starburst galaxies, however, the trends suggest flows out of the stellar disc plane (with outflow 1/2-opening angle > 45°) instead of from the nucleus: the wind velocity decreases as the disc becomes more edge-on, and the outflow fraction, constant at $\sim 90~{{\ \rm per\ cent}}$ for disc inclinations i < 45°, steadily decreases from $\sim 90~{{\ \rm per\ cent}}$ to 20 per cent for i > 45°.

Radio survey of the stellar population in the infrared dark cloud G14.225-0.506

Context. The infrared dark cloud (IRDC) G14.225-0.506 is part of the extended and massive molecular cloud located to the southwest of the H II region M17. The cloud is associated with a network of filaments, which result in two different dense hubs, as well as with several signposts of star formation activity and a rich population of protostars and young stellar objects (YSOs). Aims. The aim of this work is to study the centimeter continuum emission in order to characterize the stellar population in both regions, as well as to study the evolutionary sequence across the IRDC G14.225-0.506. Methods. We performed deep (~ 1.5–3 μJy) radio continuum observations at 6 and 3.6 cm toward the IRDC G14.225-0.506 using the Karl G. Jansky Very Large Array (VLA) in its most extended A configuration (~0.3″). Data at both C and X bands were imaged using the same (u,v) range in order to derive spectral indices. We have also made use of observations taken during different days to study the presence of variability at short timescales toward the detected sources. Results. We detected a total of 66 sources, 32 in the northern region G14.2-N and 34 in the southern region G14.2-S. Ten of the sources are found to be variable, with three located in G14.2-N and seven in G14.2-S. Based on their spectral index, the emission in G14.2-N is mainly dominated by nonthermal sources while G14.2-S contains more thermal emitters. Approximately 75% of the sources present a counterpart at other wavelengths. When considering the inner 0.4 pc region around the center of each hub, the number of infrared (IR) sources in G14.2-N is larger than in G14.2-S by a factor of 4. We also studied the relation between the radio luminosity and the bolometric luminosity, finding that the thermal emission of the studied sources is compatible with thermal radio jets. For our sources with X-ray counterparts, the nonthermal emitters follow a Güdel-Benz relation with κ = 0.03, as previously suggested for other similar regions. Conclusions. We found similar levels of fragmentation between G14.2-N and G14.2-S, suggesting that both regions are most likely twin hubs. The nonthermal emission found in the less evolved objects, mainly coming from G14.2-N, suggests that G14.2-N may be composed of more massive YSOs as well as being in a more advanced evolutionary stage, consistent with the “filament-halo” gradient in age and mass from previous works. Overall, our results confirm a wider evolutionary sequence from the southwest to northeast starting in G14.2-S as the youngest part, followed by G14.2-N, and ending with the most evolved region M17.

Research Progress on hCNT3 Structure/Function and Nucleoside Anticancer Drugs.

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na+ and H+ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.

Sequence Evolution and Copy Number Variation in HINTW

Sequence Evolution and Copy Number Variation in HINTW

A Tool to Teach Evolution of Protein Sequences and Structures

Computer modeling and protein structure visualization tools are effective and engaging ways of presenting various molecular biology concepts to high school and college students. Here, we describe a series of activities and exercises that use online bioinformatics databases and programs to search for and obtain protein sequence and structure data and use it to build homology models of proteins. Exercises in homology modeling can serve the pedagogical purpose of introducing and illustrating the concept of homology within gene and protein families, which results in conservation of the 3D structures of proteins and allows us to predict structures when experimental data are not available.

An epistatic model of sequence evolution generates evolved sequences with in vivo antibiotic functionality

An epistatic model of sequence evolution generates evolved sequences with in vivo antibiotic functionality

Positive Selection Drives cis-regulatory Evolution Across the Threespine Stickleback Y Chromosome.

Allele-specific gene expression evolves rapidly on heteromorphic sex chromosomes. Over time, the accumulation of mutations on the Y chromosome leads to widespread loss of gametolog expression, relative to the X chromosome. It remains unclear if expression evolution on degrading Y chromosomes is primarily driven by mutations that accumulate through processes of selective interference, or if positive selection can also favor the down-regulation of coding regions on the Y chromosome that contain deleterious mutations. Identifying the relative rates of cis-regulatory sequence evolution across Y chromosomes has been challenging due to the limited number of reference assemblies. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an excellent model to identify how regulatory mutations accumulate on Y chromosomes due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across 3 differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions on the Y chromosome exhibited elevated substitution rates and decreased polymorphism when compared to nonfunctional sites, like intergenic regions and synonymous sites. This suggests that many cis-regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked cis-regulatory regions exhibit signs of positive selection quickly after the suppression of recombination and allow comparisons with recent theoretical models that suggest the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome.

In vivo functional phenotypes from a computational epistatic model of evolution

Computational models of evolution are valuable for understanding the dynamics of sequence variation, to infer phylogenetic relationships or potential evolutionary pathways and for biomedical and industrial applications. Despite these benefits, few have validated their propensities to generate outputs with in vivo functionality, which would enhance their value as accurate and interpretable evolutionary algorithms. We demonstrate the power of epistasis inferred from natural protein families to evolve sequence variants in an algorithm we developed called sequence evolution with epistatic contributions (SEEC). Utilizing the Hamiltonian of the joint probability of sequences in the family as fitness metric, we sampled and experimentally tested for in vivo [Formula: see text]-lactamase activity in Escherichia coli TEM-1 variants. These evolved proteins can have dozens of mutations dispersed across the structure while preserving sites essential for both catalysis and interactions. Remarkably, these variants retain family-like functionality while being more active than their wild-type predecessor. We found that depending on the inference method used to generate the epistatic constraints, different parameters simulate diverse selection strengths. Under weaker selection, local Hamiltonian fluctuations reliably predict relative changes to variant fitness, recapitulating neutral evolution. SEEC has the potential to explore the dynamics of neofunctionalization, characterize viral fitness landscapes, and facilitate vaccine development.

A Narrative Review of Current Knowledge on Cutaneous Melanoma.

Cutaneous melanoma is a public health problem. Efforts to reduce its incidence have failed, as it continues to increase. In recent years, many risk factors have been identified. Numerous diagnostic systems exist that greatly assist in early clinical diagnosis. The histopathological aspect illustrates the grim nature of these cancers. Currently, pathogenic pathways and the tumor microclimate are key to the development of therapeutic methods. Revolutionary therapies like targeted therapy and immune checkpoint inhibitors are starting to replace traditional therapeutic methods. Targeted therapy aims at a specific molecule in the pathogenic chain to block it, stopping cell growth and dissemination. The main function of immune checkpoint inhibitors is to boost cellular immunity in order to combat cancer cells. Unfortunately, these therapies have different rates of effectiveness and side effects, and cannot be applied to all patients. These shortcomings are the basis of increased incidence and mortality rates. This study covers all stages of the evolutionary sequence of melanoma. With all these data in front of us, we see the need for new research efforts directed at therapies that will bring greater benefits in terms of patient survival and prognosis, with fewer adverse effects.

Comparative Phylogenetic Analysis of Ancient Korean Tea "Hadong Cheon-Nyeon Cha (Camellia sinensis var. sinensis)" Using Complete Chloroplast Genome Sequences.

Wild teas are valuable genetic resources for studying evolution and breeding. Here, we report the complete chloroplast genome of the ancient Korean tea 'Hadong Cheon-nyeon Cha' (C. sinensis var. sinensis), which is known as the oldest tea tree in Korea. This study determined seven Camellia sinensis var. sinenesis, including Hadong Cheon-nyeon Cha (HCNC) chloroplast genome sequences, using Illumina sequencing technology via de novo assembly. The chloroplast genome sizes ranged from 157,019 to 157,114 bp and were organized into quadripartite regions with the typical chloroplast genomes. Further, differences in SNPs and InDels were detected across the seven chloroplast genomes through variance analysis. Principal component and phylogenetic analysis suggested that regional constraints, rather than functional constraints, strongly affected the sequence evolution of the cp genomes in this study. These genomic resources provide evolutionary insight into Korean tea plant cultivars and lay the foundation for a better understanding of the ancient Korean tea plant HCNC.

Remote seizures and drug-resistant epilepsy after a first status epilepticus in adults.

Long-term consequences after status epilepticus (SE) represent an unsettled issue. We investigated the incidence of remote unprovoked seizures (RS) and drug-resistant epilepsy (DRE) in a cohort of first-ever SE survivors. A retrospective, observational, and monocentric study was conducted on adult patients (age ≥ 14 years) with first SE who were consecutively admitted to the Modena Academic Hospital, Italy (September 2013-March 2022). Kaplan-Meier survival analyses were used to calculate the probability of seizure freedom following the index event, whereas Cox proportional hazard regression models were used to identify outcome predictors. A total of 279 patients were included, 57 of whom (20.4%) developed RS (mean follow-up = 32.4 months). Cumulative probability of seizure freedom was 85%, 78%, and 68% respectively at 12 months, 2 years, and 5 years. In 45 of 57 patients (81%), the first relapse occurred within 2 years after SE. The risk of RS was higher in the case of structural brain damage (hazard ratio [HR] = 2.1, 95% confidence interval [CI] = 1.06-4.01), progressive symptomatic etiology (HR = 2.7, 95% CI = 1.44-5.16), and occurrence of nonconvulsive evolution in the semiological sequence of SE (HR = 2.9, 95% CI = 1.37-6.37). Eighteen of 57 patients (32%) developed DRE; the risk was higher in the case of super-refractory (p = 0.006) and non-convulsive SE evolution (p = 0.008). The overall risk of RS was moderate, temporally confined within 2 years after the index event, and driven by specific etiologies and SE semiology. Treatment super-refractoriness and non-convulsive SE evolution were associated with DRE development.

TinyPredNet: A Lightweight Framework for Satellite Image Sequence Prediction

Satellite image sequence prediction aims to precisely infer future satellite image frames with historical observations, which is a significant and challenging dense prediction task. Though existing deep learning models deliver promising performance for satellite image sequence prediction, the methods suffer from quite expensive training costs, especially in training time and GPU memory demand, due to the inefficiently modeling for temporal variations. This issue seriously limits the lightweight application in satellites such as space-borne forecast models. In this article, we propose a lightweight prediction framework TinyPredNet for satellite image sequence prediction, in which a spatial encoder and decoder model the intra-frame appearance features and a temporal translator captures inter-frame motion patterns. To efficiently model the temporal evolution of satellite image sequences, we carefully design a multi-scale temporal-cascaded structure and a channel attention-gated structure in the temporal translator. Comprehensive experiments are conducted on FengYun-4A (FY-4A) satellite dataset, which show that the proposed framework achieves very competitive performance with much lower computation cost compared to state-of-the-art methods. In addition, corresponding interpretability experiments are conducted to show how our designed structures work. We believe the proposed method can serve as a solid lightweight baseline for satellite image sequence prediction.

Sensing Levofloxacin with an RNA Aptamer as a Bioreceptor.

To combat the growing threat of antibiotic resistance, environmental testing for antibiotic contamination is gaining an increasing role. This study aims to develop an easy-to-use assay for the detection of the fluoroquinolone antibiotic levofloxacin. Levofloxacin is used in human and veterinary medicine and has been detected in wastewater and river water. An RNA aptamer against levofloxacin was selected using RNA Capture-SELEX. The 73 nt long aptamer folds into three stems with a central three-way junction. It binds levofloxacin with a Kd of 6 µM and discriminates the closely related compound ciprofloxacin. Furthermore, the selection process was analyzed using a next-generation sequencing approach to better understand the sequence evolution throughout the selection. The aptamer was used as a bioreceptor for the development of a lateral flow assay. The biosensor exploited the innate characteristic of RNA Capture-SELEX to select aptamers that displace a complementary DNA oligonucleotide upon ligand binding. The lateral flow assay achieved a limit of visual detection of 100 µM. While the sensitivity of this assay constrains its immediate use in environmental testing, the present study can serve as a template for the selection of RNA aptamer-based biosensors.

Strong mitonuclear discordance in the phylogeny of Neodermata and evolutionary rates of Polyopisthocotylea

The genomic evolution of Polyopisthocotylea remains poorly understood in comparison to the remaining three classes of Neodermata: Monopisthocotylea, Cestoda, and Trematoda. Moreover, the evolutionary sequence of major events in the phylogeny of Neodermata remains unresolved. Herein we sequenced the mitogenome and transcriptome of the polyopisthocotylean Diplorchis sp., and conducted comparative evolutionary analyses using nuclear (nDNA) and mitochondrial (mtDNA) genomic datasets of Neodermata. We found strong mitonuclear discordance in the phylogeny of Neodermata. Polyopisthocotylea exhibited striking mitonuclear discordance in relative evolutionary rates: the fastest-evolving mtDNA in Neodermata and a comparatively slowly-evolving nDNA genome. This was largely attributable to its very long stem branch in mtDNA topologies, not exhibited by the nDNA data. We found indications that the fast evolution of mitochondrial genomes of Polyopisthocotylea may be driven both by relaxed purifying selection pressures and elevated levels of directional selection. We identified mitochondria-associated genes encoded in the nuclear genome: they exhibited unique evolutionary rates, but not correlated with the evolutionary rate of mtDNA, and there is no evidence for compensatory evolution (they evolved slower than the rest of the genome). Finally, there appears to exist an exceptionally large (≈6.3 kb) nuclear mitochondrial DNA segment (numt) in the nuclear genome of newly sequenced Diplorchis sp. A 3′-end segment of the 16S rRNA gene encoded by the numt was expressed, suggesting that this gene acquired novel, regulatory functions after the transposition to the nuclear genome. In conclusion, Polyopisthocotylea appears to be the lineage with the fastest-evolving mtDNA sequences among all of Bilateria, but most of the substitutions were accumulated deep in the evolutionary history of this lineage. As the nuclear genome does not exhibit a similar pattern, the circumstances underpinning this evolutionary phenomenon remain a mystery.

Technical and tactical evolution of the offensive team sequences in LaLiga between 2008 and 2021. Is Spanish football now a more associative game?

The aim of this investigation was to study the technical and tactical evolution of the offensive team sequences in the Spanish football teams from 2008/09 to 2020/21. A comparative analysis including twelve variables related to the development of offensive sequences in 4940 matches was performed from 2008/09 to 2020/21 seasons of the Spanish professional football league (LaLiga). All match observations were recorded using a validated video tracking system. Multilevel linear mixed models were used to examine the differences across seasons, considering the effects of contextual variables. The number of passes per sequence (2.4 [CI: 2.2-2.5] vs 3.2 [CI: 3.0-3.4]; +33.3%), the passing accuracy (72.1 [CI: 70.6-73.5] vs 76.9 [CI: 75.4-78.3]%; +6.8%) and the average duration of the team sequences (6.4 [CI: 5.9-6.8] vs 8.3 [CI: 7.8-8.7] seconds; +25.76%) showed a small increasing trend over the seasons (P < 0.05). In contrast, variables such as the direct speed of progression (2.2 [CI: 2.1-2.3] vs 1.6 [CI: 1.5-1.7] metres/second; -24.5%), key passes (8.1 [CI: 7.6-8.5] vs 6.8 [CI: 6.3-7.2]; -15.8%), and the sequences that ended in the attacking third (64.8 [CI: 62,7-66.8] vs 57.1 [CI: 55.1-59.2]; -11.7%) or in a shot (13.0 [CI: 12.4-13.6] vs 10.2 [CI: 9.6-10.8]; -21.6%) showed a small decreasing trend from 2008/09 to 2020/21 (P < 0.05). Spanish professional football teams slightly evolved technically and tactically towards a more associative style of play that includes longer passing sequences. This evolution also involved a decreasing speed of progression and fewer technical actions such as through balls, key passes and shots.

In silico analysis of Apostasia wallichii (Apostasioideae) and Ludisia discolor (Orchidoideae) orchids reveals different repeats composition despite the same genome size.

Repetitive sequences can lead to variation in DNA quantity and composition among species. The Orchidaceae, the largest angiosperm family, is divided into five subfamilies, with Apostasioideae as the basal group and Orchidoideae and Epidendroideae showing high diversification rates. Despite their different evolutionary paths, some species in these groups have similar nuclear DNA content. This study focuses on one example to understand the dynamics of major repetitive DNAs in the nucleus. We used Next-Generation Sequencing (NGS) data from Apostasia wallichii (Apostasioideae) and Ludisia discolor (Orchidoideae) to identify and quantify the most abundant repeats. The repetitive fraction varied in abundance (27.5% in L. discolor and 60.6% in A. wallichii) and composition, with LTR retrotransposons of different lineages being the most abundant repeats in each species. Satellite DNAs showed varying organization and abundance. Despite the unbalanced ratio between single-copy and repetitive DNA sequences, the two species had the same genome size, possibly due to the elimination of non-essential genes. This phenomenon has been observed in other Apostasia and likely led to the proliferation of transposable elements in A. wallichii. Deep genome information in the future will aid in understanding the contraction/expansion of gene families and the evolution of sequences in these genomes.

Treatment trajectories and outcomes in inflammatory bowel disease: a tertiary single-centre experience.

There is an increasing diversification in the treatment landscape for inflammatory bowel diseases (IBD) leading to therapeutic challenges that can only incompletely be covered by prospective randomized double-blind trials. Real-world observations are therefore an important tool to provide insights into therapeutic strategies. To describe the real-world treatment algorithms in an IBD referral centre. Single-centre retrospective cohort study. We retrospectively analysed prospectively collected data on treatment sequences and outcomes from 502 patients with Crohn's disease (CD) and ulcerative colitis (UC) treated with infliximab, adalimumab, vedolizumab or ustekinumab at a large German tertiary referral centre. Treatment decisions correlated to baseline patient characteristics. Over time, infliximab continued to be the preferred first-line option in CD and UC, although ustekinumab and vedolizumab, respectively, became increasingly important choices. Remission rates decreased with the advancement of therapy lines. We provide insights into the evolution of tertiary centre real-world treatment sequences that might - together with other observations - help to guide the selection of therapies in IBD. Our data also strongly underscore the unmet need for biomarkers supporting treatment decisions. None.