Integration of genomic and ecological methods inform management of an undescribed, yet highly exploited, sardine species.

Assessing genetic diversity within species is key for conservation strategies in the context of human-induced biotic changes. This is important in marine systems, where many species remain undescribed while being overfished, and conflicts between resource-users and conservation agencies are common. Combining niche modelling with population genomics can contribute to resolving those conflicts by identifying management units and understanding how past climatic cycles resulted in current patterns of genetic diversity. We addressed these issues on an undescribed but already overexploited species of sardine of the genus Harengula. We find that the species distribution is determined by salinity and depth, with a continuous distribution along the Brazilian mainland and two disconnected oceanic archipelagos. Genomic data indicate that such biogeographic barriers are associated with two divergent intraspecific lineages. Changes in habitat availability during the last glacial cycle led to different demographic histories among stocks. One coastal population experienced a 3.6-fold expansion, whereas an island-associated population contracted 3-fold, relative to the size of the ancestral population. Our results indicate that the island population should be managed separately from the coastal population, and that a Marine Protected Area covering part of the island population distribution can support the viability of this lineage.

Postoperative Imaging of Endometriosis.

Endometriosis is a highly prevalent disease that affects 10%-15% of women of reproductive age worldwide and is mainly associated with chronic pelvic pain and infertility. With the widespread use of imaging for the diagnosis and monitoring of endometriosis, combined with the ability of surgery to eradicate the disease and address infertility, there has been a significant increase in recent years in imaging examinations for postoperative evaluation of endometriosis. US and MRI are used not only to help diagnose and map endometriosis but also to evaluate refractory symptoms, residual lesions, and complications at posttreatment assessment. Knowledge of surgical techniques and recognition of expected postoperative imaging findings are crucial to differentiate postoperative changes from residual disease and/or recurrence. The authors discuss imaging aspects of postoperative endometriosis, with an emphasis on the imaging approach, comprehension of surgical techniques, recognition of the expected findings, possible complications, and analysis of residual disease or recurrence. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material. See the invited commentary by VanBuren in this issue. The slide presentation from the RSNA Annual Meeting is available for this article.

The annual ovarian cycle in Northern pit vipers (Bothrops atrox) using serum steroids and ultrasound of the female reproductive tract.

Knowledge on hormonal regulation of reproductive cycles in viperid snakes is still incipient, especially when it comes to females and tropical species. There is an urgent need to understand the reproduction of venomous snakes to improve assisted reproduction techniques and optimize the maintenance of these animals in captivity. With this in mind, we monitored Northern pit viper females year-round throughout different seasons via serum levels of progesterone (P4) and estradiol (E2) in conjunction with ultrasound examinations. Ovarian follicles were classified according to their size and stage of vitellogenesis in F-I and F-II (non-vitellogenic phase) or in F-III and F-IV (vitellogenic phase). During autumn and winter, five adult males were rotated among these females for reproductive pairing, which resulted in 17 copulations and 2 pregnancies in the first year and 12 copulations and 5 pregnancies in the second year. Then, we assessed changes in P4 and E2 levels according to seasons, predominant ovarian structures and the presence of embryos or eggs in the oviduct. Our findings showed high levels of E2 when a greater number of vitellogenic follicles were detected, indicating a possible influence of E2 on vitellogenesis and higher levels of P4 whenever eggs and embryos were visualized in the oviduct, implying its role in maintaining pregnancy. Descriptive analysis of the vipers' ovarian cycles revealed a greater number of vitellogenic follicles during winter, probably as a result of increases in E2; whereas pregnancies occurred predominantly in spring, under the influence of P4. The use of ultrasound images, as a minimally invasive methodology, associated with serum steroid levels has proven to be an efficient approach in the reproductive monitoring of Northern pit vipers in vivo. In addition, these data suggest that female pit vipers under human care display a seasonal reproductive cycle, despite earlier studies involving captive males of the species indicating a lack of seasonality in sperm production and quality.

Improving Bioprocess Conditions for the Production of Prodigiosin Using a Marine Serratia rubidaea Strain.

The enormous potential attributed to prodigiosin regarding its applicability as a natural pigment and pharmaceutical agent justifies the development of sound bioprocesses for its production. Using a Serratia rubidaea strain isolated from a shallow-water hydrothermal vent, optimization of the growth medium composition was carried out. After medium development, the bacterium temperature, light and oxygen needs were studied, as was growth inhibition by product concentration. The implemented changes led to a 13-fold increase in prodigiosin production in a shake flask, reaching 19.7 mg/L. The conditions allowing the highest bacterial cell growth and prodigiosin production were also tested with another marine strain: S. marcescens isolated from a tide rock pool was able to produce 15.8 mg/L of prodigiosin. The bioprocess with S. rubidaea was scaled up from 0.1 L shake flasks to 2 L bioreactors using the maintenance of the oxygen mass transfer coefficient (kLa) as the scale-up criterion. The implemented parameters in the bioreactor led to an 8-fold increase in product per biomass yield and to a final concentration of 293.1 mg/L of prodigiosin in 24 h.

Out of Asia? Expansion of Eurasian Lyme borreliosis causing genospecies display unique evolutionary trajectories.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens.

Development of Silk Fibroin Scaffolds for Vascular Repair.

Silk fibroin (SF) is a biocompatible natural protein with excellent mechanical characteristics. SF-based biomaterials can be structured using a number of techniques, allowing the tuning of materials for specific biomedical applications. In this study, SF films, porous membranes, and electrospun membranes were produced using solvent-casting, salt-leaching, and electrospinning methodologies, respectively. SF-based materials were subjected to physicochemical and biological characterizations to determine their suitability for tissue regeneration applications. Mechanical analysis showed stress-strain curves of brittle materials in films and porous membranes, while electrospun membranes featured stress-strain curves typical of ductile materials. All samples showed similar chemical composition, melting transition, hydrophobic behavior, and low cytotoxicity levels, regardless of their architecture. Finally, all of the SF-based materials promote the proliferation of human umbilical vein endothelial cells (HUVECs). These findings demonstrate the different relationship between HUVEC behavior and the SF sample's topography, which can be taken advantage of for the design of vascular implants.

Combining high pressure and electric fields towards Nannochloropsis oculata eicosapentaenoic acid-rich extracts.

Nannochloropsis oculata is naturally rich in eicosapentaenoic acid (EPA). To turn this microalga into an economically viable source for commercial applications, extraction efficiency must be achieved. Pursuing this goal, emerging technologies such as high hydrostatic pressure (HHP) and moderate electric fields (MEF) were tested, aiming to increase EPA accessibility and subsequent extraction yields. The innovative approach used in this study combined these technologies and associated tailored, less hazardous different solvent mixtures (SM) with distinct polarity indexes. Although the classical Folch SM with chloroform: methanol (PI 4.4) provided the highest yield concerning total lipids (166.4 mglipid/gbiomass), diethyl ether: ethanol (PI 3.6) presented statistically higher values in terms of EPA per biomass, corresponding to 1.3-fold increase. When SM were used in HHP and MEF, neither technology independently improved EPA extraction yields, although the sequential combination of technologies did result in 62% increment in EPA extraction. Overall, the SM and extraction methodologies tested (HHP-200 MPa, 21 °C, 15 min, followed by MEF processing at 40 °C, 15 min) enabled increased EPA extraction yields from wet N. oculata biomass. These findings are of high relevance for the food and pharmaceutical industries, providing viable alternatives to the "classical" extraction methodologies and solvents, with increased yields and lower environmental impact. KEY POINTS: • Et2O: EtOH is a less toxic and more efficient alternative to Folch solvent mixture • HHP or MEF per se was not able to significantly increase EPA extraction yield • Combinations of HHP and MEF technologies increased both lipids and EPA yields.

Best Medical Treatment in Patients with Asymptomatic Carotid Stenosis: Myth or Reality?

BACKGROUND: Approximately 1.4 million strokes/year causing about 1.1 million deaths annually occur in Europe and 10%-15% of those strokes are result of thromboembolism from a previously significant asymptomatic carotid stenosis (ACS). Medical treatment has improved considerably in the last 15 years; however, its success depends on patient compliance. The aim of our study was to evaluate, in patients with ACS, the implementation and patient adherence to best medical treatment (BMT). Additionally, we sought to determine the "real-world" incidence of cerebrovascular/coronary events in a cohort of nonoperated ACS patients and weighing this risk against surgical complications in patients with ACS undergoing surgical treatment at our Department. METHODS: Patients with ACS ≥ 60% identified by a carotid ultrasound performed at our Department were retrospectively evaluated. Patients selected to BMT were excluded if the follow-up period was inferior to 2 years, as well as patients lost in follow-up, with missing clinical information and submitted to carotid stenting. Patients' data collection was supported by hospital reporting system and data were introduced into a database created for the purpose. Statistical analysis was performed using SPSS-25 software. RESULTS: After exclusion criteria were applied, the last 120 consecutive patients (60 with ACS submitted do carotid endarterectomy and 60 with ACS under BMT) were retrospectively evaluated. Twenty one patients had ipsilateral events for more than 6 months. Most patients had hypertension (n = 107; 89%), dyslipidemia (n = 101; 84%), 40% had diabetes, 33% diagnosed coronary disease, 32% were overweight or obese, and 17% were current smokers. Blood pressure control, normal weight, statin with/without ezetimibe association, and antiaggregant therapy were only achieved in 33 patients and only 5 had additionally low-density lipoprotein levels < 70 mg/dL, hemoglobin A1c < 7%, and were nonsmokers. Of the 60 patients assigned to medical treatment, 3 (5%) had a stroke at 2 years of follow-up, which was fatal in 1 patient. Among patients submitted to carotid endarterectomy, perioperative stroke was documented in 3% of the patients, none of them disabling or fatal. CONCLUSIONS: Although some recent studies report a risk of ipsilateral stroke of only 0.34% per year in patients with ACS ≥ 50% under BMT therapy in our everyday practice strict compliance to medical treatment fails in most patients. In consequence, we think that a "one-size-fits-all" guideline policy may not be appropriate for all patients and the management of specific ACS patients may need to be individualized.

Isolation and Characterization of a Serratia rubidaea from a Shallow Water Hydrothermal Vent.

Microbial life present in the marine environment has to be able to adapt to rapidly changing and often extreme conditions. This makes these organisms a putative source of commercially interesting compounds since adaptation provides different biochemical routes from those found in their terrestrial counterparts. In this work, the goal was the identification of a marine bacterium isolated from a sample taken at a shallow water hydrothermal vent and of its red product. Genomic, lipidomic, and biochemical approaches were used simultaneously, and the bacterium was identified as Serratia rubidaea. A high-throughput screening strategy was used to assess the best physico-chemical conditions permitting both cell growth and production of the red product. The fatty acid composition of the microbial cells was studied to assess adaptation at the lipid level under stressful conditions, whilst several state-of-the-art techniques, such as DSC, FTIR, NMR, and Ultra-High Resolution Qq-Time-of-Flight mass spectrometry, were used to characterize the structure of the pigment. We hypothesize that the pigment, which could be produced by the cells up to 62 °C, is prodigiosin linked to an aliphatic compound that acts as an anchor to keep it close to the cells in the marine environment.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp) kept in captivity.

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Cytogenomic Analysis of Long-Term Epilepsy-Associated Tumors Using an Array-Based CGH Strategy.

A palette of copy number changes in long-term epilepsy-associated tumors (LEATs) have been reported, but the data are heterogeneous. To better understand the molecular basis underlying the development of LEATs, we performed array-comparative genomic hybridization analysis to investigate chromosomal imbalances across the entire genome in 8 cases of LEATs. A high number of aberrations were found in 4 patients, among which deletions predominated. Both whole-chromosome and regional abnormalities were observed, including monosomy 19, deletion of 1p, deletions of 4p, 12p, and 22q, and gain of 20p. The common altered regions are located mainly on chromosomes 19 and 4p, identifying genes potentially involved in biological processes and cellular mechanisms related to tumorigenesis. Our study highlights new genomic alterations and reinforces others previously reported, offering new molecular insights that may help in diagnosis and therapeutic decision-making.

A lizard with two tales: What diversification within Sceloporus occidentalis teaches us about species formation.

In 1859, Charles Darwin proposed that species are not fundamentally different from subspecies or the varieties from which they evolve. A century later, Dobzhansky (1958) suggested that many such lineages are ephemeral and are likely to reverse differentiation through introgression (Figure 1a); only a few evolve complete reproductive isolation and persist in sympatry. In this issue of Molecular Ecology, Bouzid et al. (2021) showed how new analytical methods, when applied to genomic data, allow us to more precisely determine whether or not species formation follows the paths outlined by Darwin and Dobzhansky (Figure 1b). The authors studied the diversification of the lizard Sceloporus occidentalis, finding a continuum of genetic interactions between the preservation of genetic identity to genetic merger, analogous to what is exemplified by ring species. In doing so, they teach us two tales about species formation: that lineages are fractal byproducts of evolutionary processes such as genetic drift and selection, and that lineages are often ephemeral and do not always progress into fully reproductively isolated taxa. Studying ephemeral lineages like those in S. occidentalis allows us to capture divergence at its earliest stages, and potentially to determine the factors that allow lineages to remain distinct despite pervasive gene flow. These lineages thus serve as a natural laboratory to address long standing hypotheses about species formation.

Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones.

The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions.

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Endogenous Fluorescent Proteins in the Mucus of an Intertidal Polychaeta: Clues for Biotechnology.

The vast ocean holds many unexplored organisms with unique adaptive features that enable them to thrive in their environment. The secretion of fluorescent proteins is one of them, with reports on the presence of such compounds in marine annelids being scarce. The intertidal Eulalia sp. is an example. The worm secretes copious amounts of mucus, that when purified and concentrated extracts, yield strong fluorescence under UV light. Emission has two main maxima, at 400 nm and at 500 nm, with the latter responsible for the blue-greenish fluorescence. Combining proteomics and transcriptomics techniques, we identified ubiquitin, peroxiredoxin, and 14-3-3 protein as key elements in the mucus. Fluorescence was found to be mainly modulated by redox status and pH, being consistently upheld in extracts prepared in Tris-HCl buffer with reducing agent at pH 7 and excited at 330 nm. One of the proteins associated with the fluorescent signal was localized in secretory cells in the pharynx. The results indicate that the secretion of fluorescent proteinaceous complexes can be an important defense against UV for this dweller. Additionally, the internalization of fluorescent complexes by ovarian cancer cells and modulation of fluorescence of redox status bears important considerations for biotechnological application of mucus components as markers.

Reproducibility of MRI Features of Uterine Leiomyomas: A Study on Interobserver Agreement and Inter-Method Agreement With Surgery.

PURPOSE: To evaluate interobserver agreement in the interpretation of different MRI features of uterine leiomyomas (UL) according to observers' experience, and to assess the inter-method reproducibility (MRI versus surgery) regarding the International Federation of Gynecology and Obstetrics (FIGO) classification. METHODS: Retrospective study including UL patients who underwent MRI and surgical treatment. Four blinded observers (2 vs >10 years of experience) assessed UL regarding dimensions and volume; inner and outer mantles; FIGO classification; vascularization; degeneration; and diffusion-weighted imaging features. Uterine dimensions and volume were calculated. FIGO classification as ascertained by observers was compared to surgical findings. Intraclass correlation coefficient (ICC) estimates were used for interobserver comparison of numerical variables, and kappa statistic for categorical variables. RESULTS: Thirty-five patients (26y-73y) with 61 UL were included in the interobserver analyses, and 31 patients (54 UL) had available data allowing retrospective surgical FIGO classification for assessment of inter-method reproducibility. Both groups of observers had good to excellent agreement in assessing UL (ICC = 0.980-0.994) and uterine volumes (ICC = 0.857-0.914), mantles measurement (ICC = 0.797-0.920), and apparent diffusion coefficient calculation (ICC = 0.787-0.883). There was substantial agreement for both groups regarding FIGO classification (κ = 0.645-0.767). Vascularization, degeneration and restricted diffusion had lower agreement, varying from reasonable to moderate. Inter-method agreement was reasonable (κ = 0.341-0.395). CONCLUSIONS: Interobserver agreement of MRI for UL was higher for quantitative than qualitative features, with a little impact of observers' experience for most features. MRI agreement with surgery was reasonable. Further efforts should be taken to improve interobserver and inter-method reproducibility for MRI in this scenario.

Recovery from hybrid breakdown reveals a complex genetic architecture of mitonuclear incompatibilities.

Reproductive isolation is often achieved when genes that are neutral or beneficial in their genomic background become functionally incompatible in a foreign genomic background, causing inviability, sterility or other forms of low fitness in hybrids. Recent studies suggest that mitonuclear interactions are among the initial incompatibilities to evolve at early stages of population divergence across taxa. Yet, the genomic architecture of mitonuclear incompatibilities has rarely been elucidated. We employ an experimental evolution approach starting with low-fitness F2 interpopulation hybrids of the copepod Tigriopus californicus, in which frequencies of compatible and incompatible nuclear alleles change in response to an alternative mitochondrial background. After about nine generations, we observe a generalized increase in population size and in survivorship, suggesting efficiency of selection against maladaptive phenotypes. Whole genome sequencing of evolved populations showed some consistent allele frequency changes across three replicates of each reciprocal cross, but markedly different patterns between mitochondrial backgrounds. In only a few regions (~6.5% of the genome), the same parental allele was overrepresented irrespective of the mitochondrial background. About 33% of the genome showed allele frequency changes consistent with divergent selection, with the location of these genomic regions strongly differing between mitochondrial backgrounds. In 87% and 89% of these genomic regions, the dominant nuclear allele matched the associated mitochondrial background, consistent with mitonuclear co-adaptation. These results suggest that mitonuclear incompatibilities have a complex polygenic architecture that differs between populations, potentially generating genome-wide barriers to gene flow between closely related taxa.

Genomic methods reveal independent demographic histories despite strong morphological conservatism in fish species.

Human overexploitation of natural resources has placed conservation and management as one of the most pressing challenges in modern societies, especially in regards to highly vulnerable marine ecosystems. In this context, cryptic species are particularly challenging to conserve because they are hard to distinguish based on morphology alone, and thus it is often unclear how many species coexist in sympatry, what are their phylogenetic relationships and their demographic history. We answer these questions using morphologically similar species of the genus Mugil that are sympatric in the largest coastal Marine Protected Area in the Tropical Southwestern Atlantic marine province. Using a sub-representation of the genome, we show that individuals are assigned to five highly differentiated genetic clusters that are coincident with five mitochondrial lineages, but discordant with morphological information, supporting the existence of five species with conserved morphology in this region. A lack of admixed individuals is consistent with strong genetic isolation between sympatric species, but the most likely species tree suggests that in one case speciation has occurred in the presence of interspecific gene flow. Patterns of genetic diversity within species suggest that effective population sizes differ up to two-fold, probably reflecting differences in the magnitude of population expansions since species formation. Together, our results show that strong morphologic conservatism in marine environments can lead to species that are difficult to distinguish morphologically but that are characterized by an independent evolutionary history, and thus that deserve species-specific management strategies.

Prognostic Value of Charlson Comorbidity Index in Acute Embolic Lower Limb Ischaemia Patients.

INTRODUCTION: Charlson Comorbidity Index (CCI) is commonly used in outcome studies to adjust for patient comorbid conditions but has not been specifically validated for use in acute embolic lower limb ischaemia (AELLI). OBJECTIVES: The aim of this study was to access whether a high CCI score unadjusted (CCIu) and adjusted (CCIa) by age relates to major amputation and mortality after a first episode of AELLI. METHODS: The last 100 patients presenting with the first event of AELLI submitted to embolectomy at our Vascular Surgery Department were retrospectively evaluated. Patient characteristics, pre- and post-operative period variables were collected and CCIu and CCIa calculated. Survival predictors were analyzed using Cox regression. The area under the curve of the receiver operating characteristic curves was calculated to validate and determine the discriminating ability of CCIu and CCIa in predicting amputation rate and 30-day mortality. Youden index was used to determine the critical value. Survival analysis was performed with Kaplan-Meier method and differences between survival curves were tested with Log-Rank test. A P value of <0.05 was considered statistically significant. RESULTS: The mean age was 80.03±10.776 years and the mean follow-up 19.28±7.929 months. Amputation rate was 16%, 30-day mortality 21% and long-term mortality 42%. Patients with CCIu≥3 compared with patients with CCIu<3 had higher amputation rate (37.5% vs. 1.7%; OR:35.400), 30-day mortality (47.5% vs. 3.3%; OR:35.400) and global mortality (P=0.00). Also, patients with CCIa≥6 compared with patients with CCIa<6 had higher amputation rate (34.1% vs. 1.8%; OR:28.488), 30-day mortality (47.7% vs. 0.0%) and global mortality (P=0.00). Multivariate analysis showed that both CCIu and CCIa were independent predictors of amputation rate and 30-day mortality. CONCLUSION: CCIu and CCIa have proven to be good predictors of amputation rate and survival, thus being a valuable prognostic factor in patients presenting with the first event of AELLI.