Accurate species delimitation is essential for understanding biodiversity patterns and guiding conservation, yet cryptic species with minimal morphological variation often obscure the assessment of true species diversity, making the identification of reliable diagnostic characters critical. Island radiations exemplify this challenge by concentrating endemic diversity in restricted areas, which accentuates both the urgency of conservation and the need for accurate taxonomy, while often presenting species divergences masked by subtle morphological differences. Here, we investigated patterns of morphological differentiation in Ailuronyx geckos, a genus endemic to the Seychelles archipelago, comprising deeply divergent lineages and unresolved, possibly cryptic species boundaries. We applied 3D and 2D morphometric analyses across the range (16 islands) of the three recognised species: the bronze-eyed gecko A. seychellensis, dwarf bronze gecko A. tachyscopaeus, and giant bronze gecko A. trachygaster. 3D geometric morphometric analyses of cranial and mandibular landmark data from 42 geckos revealed strong species-level differences. Ailuronyx trachygaster exhibits extreme skull sculpturing with rugose textures on up to ten cranial and three mandibular bones, while the other two species have smooth skull surfaces. Skull elongation and narrowing increased with decreasing body size from the larger A. trachygaster, via the medium-sized A. seychellensis, to the smaller A. tachyscopaeus. Analysis of body size-corrected 2D measurements of ten external morphological traits from 567 geckos revealed similar differences in head dimensions among the species, but also showed changes in mass and toe length. While we observed substantial morphological variation between islands, we did not find support for the previously identified northern and southern island group divergence within either A. seychellensis or A. tachyscopaeus. Nevertheless, we found strong support for the proposed divergence between northern and southern Mahé populations of A. tachyscopaeus, with variation in mass and head size between the two populations. We provide morphometric parameters to guide species identification within this gecko genus and highlight distinct population units that warrant further genomic investigation and prioritisation for conservation. Our findings illustrate the complexity of morphological diversification in archipelagos, where island-specific processes may outweigh regional biogeographic patterns. This underscores the need for integrative approaches to species delimitation and conservation in insular systems.

European hake is an economically important fishery resource with known declines in population abundances and biomass in the last decades. However, little is known about the effect of these abundance declines on the genetic diversity levels of the stocks. In this study, we analysed genomic data of Atlantic and Mediterranean European hake populations, representing the main fishery stocks. The results indicate that abundance declines have likely affected the levels and patterns of genetic diversity within and among the three analysed populations. The Mediterranean population shows lower genetic diversity than the Atlantic ones and can be slightly genetically differentiated from them. Results indicate significant deviations from neutral expectations for constant population sizes, and substantial population declines in all three populations. Estimates of recent and ancestral effective population size (Ne) obtained from coalescence, linkage disequilibrium and sibship methods were coherent with each other. Historical Ne estimates suggest drastic declines of more than 90% in all three populations within the last 15 generations (i.e. nearly 50 years). Positive correlations observed between historical Ne and stock parameters indicative of abundances support the historical trends of estimated Ne. Since genetic variation is essential for populations facing anthropogenically induced environmental changes, this study demonstrates the importance of population genetic estimates for planning management strategies targeting the long-term sustainability of European hake fisheries.

Distinguishing between anthropogenic introductions and natural colonizations can be complex, especially in groups that are evidently both capable natural colonizers and anthropophilic, such as some geckos of the genus Hemidactylus. However, such information is fundamental both for constructing appropriate conservation approaches, as well as to identify biogeographical patterns. Here we assessed the origins and taxonomic status of the Hemidactylus geckos found on the Îles Éparses, disjunct small islands of the western Indian Ocean located around Madagascar, using partial mitochondrial DNA sequences. Hemidactylus platycephalus was confirmed on Juan de Nova, presumed introduced from continental Africa. The H. mabouia-mercatorius complex was identified on Juan de Nova, Europa, Tromelin, and Grande Glorieuse, with H. mabouia (sensu stricto) on Juan de Nova and H. mercatorius on Europa, from where it is likely to have been introduced to Tromelin. An unnamed lineage within H. mercatorius, previously known only from the Aldabra Islands, was identified on Grande Glorieuse, and further demonstrated the unexpectedly high genetic diversity within this lineage.

EvoPPI (http://evoppi.i3s.up.pt), a meta-database for protein-protein interactions (PPI), has been upgraded (EvoPPI3) to accept new types of data, namely, PPI from patients, cell lines, and animal models, as well as data from gene modifier experiments, for nine neurodegenerative polyglutamine (polyQ) diseases caused by an abnormal expansion of the polyQ tract. The integration of the different types of data allows users to easily compare them, as here shown for Ataxin-1, the polyQ protein involved in spinocerebellar ataxia type 1 (SCA1) disease. Using all available datasets and the data here obtained for Drosophila melanogaster wt and exp Ataxin-1 mutants (also available at EvoPPI3), we show that, in humans, the Ataxin-1 network is much larger than previously thought (380 interactors), with at least 909 interactors. The functional profiling of the newly identified interactors is similar to the ones already reported in the main PPI databases. 16 out of 909 interactors are putative novel SCA1 therapeutic targets, and all but one are already being studied in the context of this disease. The 16 proteins are mainly involved in binding and catalytic activity (mainly kinase activity), functional features already thought to be important in the SCA1 disease.

Small RNA (sRNA) profiling of Extracellular Vesicles (EVs) by Next-Generation Sequencing (NGS) often delivers poor outcomes, independently of reagents, platforms or pipelines used, which contributes to poor reproducibility of studies. Here we analysed pre/post-sequencing quality controls (QC) to predict issues potentially biasing biological sRNA-sequencing results from purified human milk EVs, human and mouse EV-enriched plasma and human paraffin-embedded tissues. Although different RNA isolation protocols and NGS platforms were used in these experiments, all datasets had samples characterized by a marked removal of reads after pre-processing. The extent of read loss between individual samples within a dataset did not correlate with isolated RNA quantity or sequenced base quality. Rather, cDNA electropherograms revealed the presence of a constant peak whose intensity correlated with the degree of read loss and, remarkably, with the percentage of adapter dimers, which were found to be overrepresented sequences in high read-loss samples. The analysis through a QC pipeline, which allowed us to monitor quality parameters in a step-by-step manner, provided compelling evidence that adapter dimer contamination was the main factor causing batch effects. We concluded this study by summarising peer-reviewed published workflows that perform consistently well in avoiding adapter dimer contamination towards a greater likelihood of sequencing success.

Invasive species disrupt relations between endemics and their ecosystem and are an increasing biodiversity conservation problem. The Hemidactylus genus comprises the most successful invasive reptile species, including the worldwide-distributed Hemidactylus mabouia. In this study, we used 12S and ND2 sequences to taxonomically identify and tentatively determine the diversity and origin of these invaders in Cabo Verde while also clarifying this for several Western Indian Ocean (WIO) populations. By comparing our sequences to recently published ones, we showed, for the first time, that Cabo Verde individuals belong to the H. mabouia sensu stricto lineage and that both of its sublineages (a and b) occur there. Both haplotypes are also in Madeira, which indicates a connection between these archipelagos, possibly related to the past Portuguese trading routes. Across the WIO, results clarified the identity of many island and coastal populations, showing that this likely invasive H. mabouia lineage is widespread in the region, including northern Madagascar, with important conservation implications. Colonisation origins were difficult to access due to the wide geographical spread of these haplotypes; thus, several possible scenarios were outlined. The introduction of this species throughout western and eastern Africa may threaten endemic taxa and needs to be closely monitored.

Certain sacoglossan sea slugs can sequester and maintain photosynthetically active chloroplasts through algae feeding, a phenomenon called kleptoplasty. The period while these plastids remain active inside the slug’s body is species- and environment-dependent and can span from a few days to more than three weeks. Here we report for the first time the transcriptome of sea slug Elysia viridis (Montagu, 1804), which can maintain kleptoplasts for more than two weeks and is distributed along all the Atlantic European coastline. The obtained transcriptome of E. viridis comprised 12,884 protein-coding sequences (CDS). The shortest one was 261bp, and the longest 8,766bp; the whole transcriptome has a total length of 9.3Mb (Table S4 and Fig. S2). Analysing these CDS, we identified 9,422 different proteins, with best hits mainly from two genera: Elysia (87.2%), and Plakobranchus (11.0%) (Fig. S2); the other 2.3% corresponded to multiple genera of sea slugs and snails (Tectipleura) (Kano et al., 2016). We got the functional annotation (Gene Ontologies, GO) corresponding to 9,333 CDS: 4,755 CDS associated with 2,583 Biological Process (BP); 5,466 CDS linked to 683 Cellular Components (CC); and 6,693 related to 1,606 Molecular Functions (MF). We identified 201 CDS related to response to stress (GO:0006950) and 10 CDS associated with the regulation of response to stress (GO:0080134). Focussing on the ROS-quenching toolkit, we found 24 CDS related to oxidoreductase complex (GO:1990204) and 560 annotated with oxidoreductase activity (GO:0016491) acting in a large number of donors, e.g., CH-OH, CH=O, C=O, CH and CH2. In addition, we found 39 CDS with antioxidant activity (GO:00162099) and other CDS with ROS-quenching function: superoxide dismutase (GO:0004784), peroxidase (GO:0004601), glutathione oxidoreductase (GO:0097573) and peroxidase (GO:0004602); and thioredoxin peroxidase (GO:0008379) activity. Furthermore, we found 8 CDS related to the symbiont response (GO:0140546) and nine related to the pattern recognition receptor signalling pathway (GO:0002221).

Transfer RNA fragments (tRFs) have gene silencing effects similarly to miRNAs, can be sorted into extracellular vesicles (EVs) and are emerging as potential circulating biomarkers for cancer diagnoses. We aimed at analyzing the expression of tRFs in gastric cancer (GC) and understanding their potential as biomarkers. We explored miRNA datasets from gastric tumors and normal adjacent tissues (NATs) from TCGA repository, as well as proprietary 3D-cultured GC cell lines and corresponding EVs, in order to identify differentially represented tRFs using MINTmap and R/Bioconductor packages. Selected tRFs were validated in patient-derived EVs. We found 613 Differentially Expressed (DE)-tRFs in the TCGA dataset, of which 19 were concomitantly upregulated in TCGA gastric tumors and present in 3D cells and EVs, but barely expressed in NATs. Moreover, 20 tRFs were expressed in 3D cells and EVs and downregulated in TCGA gastric tumors. Of these 39 DE-tRFs, 9 tRFs were also detected in patient-derived EVs. Interestingly, the targets of these 9 tRFs affect neutrophil activation and degranulation, cadherin binding, focal adhesion and the cell-substrate junction, highlighting these pathways as major targets of EV-mediated crosstalk with the tumor microenvironment. Furthermore, as they are present in four distinct GC datasets and can be detected even in low quality patient-derived EV samples, they hold promise as GC biomarkers. By repurposing already available NGS data, we could identify and cross-validate a set of tRFs holding potential as GC diagnosis biomarkers.

The understanding of the molecular basis of cellular processes and ultimately disease, requires knowledge on protein structures, interactions, and functions. Protein–protein interaction data (PPI) is available in the publicly available main PPI databases that show little overlap due to the use of different criteria. Therefore, web platforms that aggregate the data from multiple sources, such as EvoPPI ( http://evoppi.i3s.up.pt ), where the existing databases have been updated and new ones were added, as here described, and APID ( http://cicblade.dep.usal.es:8080/APID/init.action ) are useful. Still, in both EvoPPI 1.0 and 2, here presented, we have made a special effort to make it flexible in what concerns the choice of the databases to be compared. Moreover, interacting protein pairs tend to be evolutionarily conserved, and thus the information available for one species might be used to predict the incompleteness of the network in another one, and identify putative missing interactions. This approach is now available in EvoPPI 2 for Homo sapiens and the model species Mus musculus, Caenorhabditis elegans, and Drosophila melanogaster, using either Ensembl ( https://www.ensembl.org ) or DIOPT Ortholog Finder ( https://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl ) orthologies/paralogies. Moreover, since not all available PPI data is present in the main databases (e.g. PPI observed in patient tissues and mutant animal species, where PPI might be aberrant, are usually not included in the main databases, although in several studies this has been shown not to be the case), we provide the needed tools (including a Lubuntu-based virtual machine where all software is already installed and ready-to-run) to run a local EvoPPI 2 instance and create a custom database from the existing ones. This way the user can add new data for any species and from any source database, creating custom interactomes. Administrator tools are provided to help in the automatic processing and conversion of files from various sources into the custom EvoPPI database format.

The authors present the case of a 79-year-old woman who presented with chronic secretory diarrhoea and severe electrolyte imbalance. A large rectal tubulovillous adenoma was discovered, leading to surgical resection of the tumour with complete resolution of the diarrhoea and electrolyte imbalance. The diagnosis of McKittrick-Wheelock syndrome was made. We hope this case report will contribute to a greater awareness among health professionals about this clinical condition. A multidisciplinary approach is essential for the best outcome.

tRNAs are a driving force of genome evolution in Yeast and Bacteria. Their deregulation is frequently observed in tumors with Serine tRNAs being often overexpressed. This has important functional consequences, such as increased metabolism and tumor growth. In yeast, time and chemical stimulus boost alterations in the genome driven by tRNA deregulation. Therefore, we hypothesized that tRNA deregulation may contribute to the increased genome instability observed in tumors. To study the effect of tRNA deregulation in tumors, we overexpressed tRNA-Ser-AGA-2-1 in a NSCLC cell line, H460. This cell line and a Mock (control) were xenografted in nude mice and collected at 3 timepoints: T1-Naïve; T2-Treated once with cisplatin/vehicle and; T3) treated twice with cisplatin/vehicle. These tumors were characterized by WES, RNAseq and Mass Spectrometry and the data obtained was integrated. The tumor mutation burden was increased in T3 tRNASerOE tumors, regardless of treatment. Although in T1 Mock and tRNASer tumors have a similar number of variants, in T2&3, tRNASerOE tumors display two times more variants than Mock tumors regardless of treatment. Interestingly, tRNASerOE exclusive variants favor proliferation and therapy resistance, which is in line with the phenotypes observed and supported by RNAseq and proteomics data. In conclusion, tRNASerOE increases the tumor mutation burden and the variants detected favor tumor growth, proving tRNA deregulation is enough to induce adaptive mutations in the genome.

Small bowel tumours are rare, representing about 0.5% of all tumours and about 3% of gastrointestinal tract tumours. The low prevalence contrasts with the vast surface area of the small intestine, which accounts for over 90% of the surface area of the digestive tract. The frequency of small tumours decreases from proximal to distal, and therefore from the duodenum to the ileum. The histological types differ in terms of prevalence according to the affected segment, with adenocarcinoma being the most frequent in the duodenum and jejunum and carcinoid tumour in the ileum. Diagnosis is challenging due to clinical non-specificity, low prevalence and a low level of suspicion. Schwannomas are typically benign tumours that arise from Schwann's cells and are rarely found in the small intestine. It is even more rare to find them together with another histological type, namely adenocarcinoma. No cases have been reported in the literature of these lesions occurring in the small intestine simultaneously. Further studies are needed to clarify the underlying pathophysiology of these synchronous tumours. The authors present the case of an 86-year-old female patient admitted for high intestinal subocclusion, with refractory vomiting and involuntary weight loss. Two synchronous lesions in the digestive tract were identified: an adenocarcinoma in the duodenum and a schwannoma in the ileum. The patient underwent surgical resection of both lesions. A high level of suspicion combined with a multidisciplinary approach is necessary for timely diagnosis and surgical resolution.LEARNING POINTS:Small bowel neoplasms are rare and clinically non-specific; in addition, diagnosis is difficult due to imaging artifacts and tumour inaccessibility for biopsy for definitive histological diagnosis.Gastrointestinal schwannomas are rare and the pathophysiology of synchrony with other histological subtypes remains to be clarified.A multidisciplinary approach from the beginning is important for a timely diagnosis and better outcome.

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.

The incidence of diabetes on the worldwide population has tripled in the past 5 decades. While drug-based therapies are valuable strategies to treat and ease the socio-economic burden of diabetes, nutritional strategies offer valuable alternatives to prevent and manage diabetes onset and contribute to the sustainability of health budgets. Whilst, intervention studies have shown that (poly)phenol-rich diets improve fasting glucose levels and other blood parameters, very little is known about the distribution of ingested polyphenols in circulation and the impact of diabetes on its cargo. In this study we investigate the impact of type 2 diabetes on the cargo of plasma (poly)phenols. Our results show that phenolic compounds are heterogeneously distributed in circulation though mainly transported by lipoprotein populations. We also found that diabetes has a marked effect on the phenolic content transported by VLDL resulting in the decrease in the content of flavonoids and consequently a decrease in the antioxidant capacity. In addition to the reduced bioavailability of (poly)phenol metabolites and increase of oxidative status in LDL and HDL populations in diabetes, cell-based assays show that sub-micromolar amounts of microbial (poly)phenol metabolites are able to counteract the pro-inflammatory status in glucose-challenged endothelial cells. Our findings highlight the relevance of triglyceride-rich lipoproteins in the transport and delivery of bioactive plant-based compounds to the endothelium in T2DM supporting the adoption of nutritional guidelines as an alternative strategy to drug-based therapeutic approaches.

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity, favouring tumour growth. We developed an EMT model by on and off exposure of epithelial EpH4 cells (E-cells) to TGFβ1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia by using RNA sequencing, immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays as well as cell xenografts in nude mice. Phenotypic reverted epithelial cells (RE-cells) obtained after MET induction presented epithelial morphologies and proliferation rates resembling E cells. However, the RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a uniquely heterogeneous mixture of cell subpopulations with a high self-renewal ability. RE cell heterogeneity was stably maintained for long periods after TGFβ1 removal both in vitro and in large tumours derived from the nude mice. Overall, we show that phenotypic reverted epithelial cells (RE cells) do not return to the molecular and functional epithelial state and present mesenchymal features related to aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cell reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour growth-promoting factor in vivo.

2 discontinuous exposure to TGFβ1 promotes tumour 3 growth 4 Mafalda Santos1,2,#, Marta Ferreira1,2,#, Patrícia Oliveira1,2, Nuno Mendes 1,2, Ana André1,2, André 5 F. Vieira1,2, Joana B. Nunes 1,2, Joana Carvalho1,2, Sara Rocha1,2, Mafalda Azevedo2, Daniel 6 Ferreira1,2,3, Inês Reis2, João Vinagre 1,2, Joana Paredes 1,2,5, Alireza Heravi-Moussavi4, Jorge Lima 7 1,2,5, Valdemar Máximo 1,2,5, Angela Burleigh4, Calvin Roskelley4, Maria de Fátima Carneiro 1,2,5, 8 David Huntsman4, Carla Oliveira 1,2,5*

Unravelling when divergent lineages constitute distinct species can be challenging, particularly in complex scenarios combining cryptic diversity and phylogenetic discordances between different types of molecular markers. Combining a phylogenetic approach with the study of contact zones can help to overcome such difficulties. The Podarcis hispanicus species complex has proven to be prosperous in independent evolutionary units, sometimes associated with cryptic diversity. Previous studies have revealed that one of the species of this complex, P. guadarramae, comprises two deeply divergent yet morphologically indistinguishable evolutionary units, currently regarded as subspecies (P. g. guadarramae and P. g. lusitanicus). In this study we used molecular data to address the systematics of the two lineages of Podarcis guadarramae and the closely related P. bocagei. Firstly, we reconstructed the species tree of these three and two additional taxa based on 30 nuclear loci using the multispecies coalescent with and without gene flow. Secondly, we used SNPs obtained from RADseq data to analyze the population structure across the distribution limits P. g. lusitanicus and P. g. guadarramae, and for comparison, a contact zone between P. bocagei and P. g. lusitanicus. Nuclear phylogenetic relationships between these three taxa are clearly difficult to determine due to the influence of gene flow, but our results give little support to the monophyly of P. guadarramae, potentially due to a nearly simultaneous divergence between them. Genetic structure and geographic cline analysis revealed that the two lineages of P. guadarramae replace each other abruptly across the sampled region and that gene flow is geographically restricted, implying the existence of strong reproductive isolation. Podarcis bocagei and P. g. lusitanicus show a similar degree of genetic differentiation and reproductive isolation, with very low levels of admixture in syntopy. These results support that all three forms are equally differentiated and reproductively isolated. In consequence, we conclude that the two former subspecies of Podarcis guadarramae constitute valid, yet cryptic species, that should be referred to as P. lusitanicus and P. guadarramae.

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.