Context Understanding how landscape change affects wildlife habitat involves integrative approaches that consider landscape structure at different scales, target species, and habitat requirements. Objectives We assessed the effects of land use and land cover (LULC) change on the availability and spatial configuration of potentially suitable habitat for three threatened large carnivores in the Iberian Peninsula: the Iberian wolf ( Canis lupus ), brown bear ( Ursus arctos ), and Iberian lynx ( Lynx pardinus ). Methods Habitat suitability was modeled for presence and breeding requirements for 1990, 2006 and 2018, and for two 2060 scenarios (rural abandonment and agricultural intensification), projected using an artificial neural network–cellular automata approach. Fragmentation was assessed via density and clustering metrics within moving windows scaled to species home ranges. Results The Iberian wolf held the largest share of suitable presence habitat but showed markedly lower connectivity for breeding habitat, a critical bottleneck. In the northwestern peninsula, brown bear habitat clustering declined despite population expansion, suggesting a temporary decoupling between demographic recovery and habitat deterioration. The Iberian lynx exhibited the highest connectivity for breeding habitat, reflecting the continuity of Mediterranean scrublands. At the Iberian scale, habitat availability and configuration remained stable between 1990 and 2018, though regional analysis revealed stronger local fragmentation. Habitat availability and connectivity are projected to improve under rural abandonment, but agricultural intensification would reduce availability and increase fragmentation, with severe consequences for bear and lynx. Conclusions Landscape change, in its composition, structure, and trajectory, threatens large carnivore habitat in the Iberian Peninsula, particularly under agricultural intensification.

Abstract Mediterranean forests have evolved over millennia under the combined influence of long-term climatic variability, which, together with natural and human disturbances, resulted in high heterogeneity of species composition and community structure. However, ongoing climate change, predicted to be particularly severe in the Mediterranean region, may threaten these forests and their biodiversity. In this work, we explore future trajectories of Iberian forests under climate change scenarios. We focus on three oak species predominantly associated with acidic substrates, Quercus suber L., Q. pyrenaica Willd. and Q. robur L., which dominate climax native forests with their optimal distribution ranges. To assess potential species turnover among focal species, we developed high-resolution spatial suitability models to project future habitat suitability for each species in the mid- and late-21st century under two climate change scenarios (SSP3-7.0 and SSP5-8.5). Results indicate different responses to climate change across species, reflecting distinct ecological thresholds, but all show predominant northward (latitudinal) and upward (altitudinal) range shifts. Projections indicate potential changes in the dominant forest species floristic composition of the Iberian oak forests’ tree layer in the future, marked by species turnover, with drought-tolerant species replacing moisture-dependent oaks across significant areas along existing environmental gradients. It is especially evident in turnover from Q. pyrenaica to Q. suber, particularly at current southern fringes and on inland areas of the Iberian Peninsula. Forest management must incorporate these projected changes through adaptive strategies, aiming to enhance forest persistence and resilience.

Since deep-diving cetaceans are one of the most understudied groups of marine mammals, basic knowledge like their distribution and abundance are generally lacking. Here we provide baseline and updated abundance estimates for the majority of the deep diving species found in the Macaronesia archipelagos, in the North-East Atlantic. Ship-board surveys were conducted in the summer and fall (July 2017-September 2018) in the Azores, Madeira, and the Canary Islands. The most abundant species was the short-finned pilot whale in the Canary Islands (0.088 animals/km2) and Madeira (0.047 animals/km2) and the Sowerby’s beaked whale in the Azores (0.017 animals/km2), while the most abundant beaked whale in the Canary Islands (0.002 animals/km2) and Madeira (0.001 animals/km 2) was the Cuvier’s beaked whale. Sperm whales were more abundant in the Azores (0.008 animals/km 2) than Madeira (0.006 animals/km 2). These surveys provided information that might be used to support current conservation mechanisms, such as the Marine Strategy Framework Directive or the Habitat Directive. Our findings also corroborated the idea that the Azores, Madeira, and the Canary Islands are open ocean insular habitats which are really important habitat for deep-diving species.

The genetic modification of human T cells to express chimeric antigen receptors (CAR-T cells) has revolutionized cancer immunotherapy by redirecting their cytotoxicity towards specific tumor antigens. While CAR-T cell therapies have demonstrated remarkable success in hematological malignancies, their translation to solid tumors remains limited by several challenges. These include the lack of exclusive tumor antigens and intrinsic tumor heterogeneity, which contribute to suboptimal targeting and increase the risk of on-target, off-tumor effects. Additionally, solid tumors present a complex and hostile tumor microenvironment (TME), characterized by multiple physical barriers and immunosuppressive mechanisms that severely hinder CAR-T cells trafficking, persistence, and anti-tumor activity. A deeper understanding of these obstacles has fueled the development of next-generation CAR designs equipped with advanced synthetic biology approaches. Improved antigen specificity with logic-gated systems, multiple-input CAR designs, co-expression of cytokine receptors, armored CARs, and engineered resistance to immunosuppressive cues in the form of chimeric switch or dominant negative receptors have emerged in response. In this context, this review provides a stepwise and comparative overview of the major biological and structural challenges limiting CAR T-cells efficacy in solid tumors. It critically discusses the innovative CAR constructs developed to overcome each of these obstacles - from antigen selection to trafficking and TME remodeling - offering a forward-looking framework to guide future research and accelerate the translation of CAR-T therapies beyond blood cancers.

Abstract Submediterranean marcescent oak forests form a climatic ecotone highly exposed to increasing aridity across the Mediterranean Basin. Understanding how these vulnerable taxa were affected by past climatic shifts can help contextualize their sensitivity to ongoing changes. Here we used ensemble Species Distribution Models (SDMs) to infer the distribution dynamics of eight marcescent oak species from the Heinrich Stadial (~ 17 ka) to the present, and to explore their potential future trajectories for 2070 and 2100 under three SSP scenarios. Models calibrated with 12,450 filtered occurrences and high-resolution paleoclimate and CHELSA datasets performed well (AUC > 0.97), with precipitation and temperature seasonality emerging as key predictors. Hindcasts revealed contrasting east–west Quaternary histories, including episodes of expansion, contraction and partial stability linked to abrupt climate transitions such as the Heinrich Stadial and Younger Dryas. Future projections indicate widespread northward shifts and substantial suitability losses, especially under SSP5-8.5, with pronounced impacts on narrowly distributed taxa. By comparing past-to-present and present-to-future range shifts, we identify temporal coherence in species responses, showing that taxa with strong historical fluctuations tend to exhibit larger projected changes. Integrating past range dynamics provides an essential ecological baseline to interpret species-specific sensitivity and regional asymmetries. Our results refine the identification of potential climatic refugia and high-risk zones, offering a framework to prioritize conservation strategies for transitional oak forests in a rapidly warming Mediterranean Basin.

Efficient lentiviral (LV) transduction is a cornerstone of CAR-T manufacturing, yet performance is often construct-specific and highly sensitive to production and delivery parameters. We developed a stepwise optimization workflow using an underperforming anti-FITC-CAR in Jurkat E6-1 cells and validated generalizability with a well-performing HER2 CAR (pHR_SFFv_4D5-WT-Highest), followed by translational testing in primary PBMCs. The strategy sequentially tuned LV concentration, brief agitation during transduction, packaging system, DNA input balance, and addition of a transduction enhancer, with outcomes quantified by flow cytometry (tdTomato and HA or c-myc tags). Concentrated supernatants and a short 2-h shaking step improved signal definition and yield; incorporating an alternative packaging plasmid and a modest DNA rebalance further increased performance. With a low-dose enhancer, Jurkat transduction with the anti-FITC-CAR arose from ∼1% to ∼40–50% tdTomato+HA+ cells (∼5–50-fold improvement, 96 h). The comparator HER2 construct—already efficient—also benefited, increasing from ∼76 to ∼88%, indicating the workflow’s utility even for high-baseline vectors. In PBMCs, the same conditions achieved ∼10% transduction at 96 h, consistent with the greater refractoriness of primary T cells and highlighting avenues for future gains via complementary steps. Overall, this modular, low-complexity optimization provides a reproducible template to rescue underperforming constructs and incrementally boost robust vectors, supporting more reliable lab-scale CAR-T engineering and offering a tractable starting point for primary T-cell protocols.

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer that remains an unmet medical need. Because TNBC cells do not express the most common markers of breast cancers, there is an active search for novel molecular targets in triple-negative tumors. Additionally, this subtype of breast cancer presents strong immunogenic characteristics which have been encouraging the development of immunotherapeutic approaches against the disease. In this context, nucleolin arises as a promising target for immunotherapy against TNBC. Our group has previously developed an anti-nucleolin VHH-Fc antibody capable of eliciting antibody-dependent cellular cytotoxicity (ADCC). Moreover, we constructed and characterized an antibody library, that was screened against nucleolin-overexpressing cells, originating an enriched anti-nucleolin antibody pool. In this work, a strategy to select individual clones from the pool was designed, combining NGS data with 3D modeling. Two antibodies demonstrated a significant 4.4- and 6.1-fold increase in binding to nucleolin-overexpressing and TNBC cells, and an improvement in affinity to the sub-micromolar range (0.19 µM and 83.69 nM). Additionally, an increment in 4.6- and 3.1-fold in ADCC activity against respective cell lines was observed for the M2 antibody clone. Herein, the affinity maturation strategy developed was validated and corroborated a positive, but not proportional, correlation between antibody binding, affinity, and ADCC.

Protein engineering is a powerful tool in biotechnology and the basis to create unprecedented sequences, structures, and functions. The WW domains are a family of naturally occurring proteins involved in the molecular recognition of proline-rich and phosphorylated peptide sequences with relevance in cellular processes involved in human diseases. Due to their small size, WW domains represent appealing small protein domains for protein engineering and to generate novel functions as binders to non-cognate targets. Here, we designed a synthetic protein scaffold library based on the WW prototype sequence in which the loops were extended and randomized while maintaining structural stability. Using in vitro evolution by phage display against human serum albumin (HSA), we found a lead candidate that was produced by biological and chemical means and further characterized using experimental and computational tools. As a potential application for the lead binder, it was immobilized on a matrix and used to capture the target HSA. Overall, this work shows the versatility of WW domains as peptide scaffolds amenable for in vitro evolution against non-cognate targets.

The open ocean is a vast, highly connected environment, and the organisms found there have been hypothesized to represent massive, well-mixed populations. Of these, the Portuguese man-o’-war ( Physalia ) is uniquely suited to dispersal, sailing the ocean surface with a muscular crest. We tested the hypothesis of a single, panmictic Physalia population by sequencing 133 genomes, and found five distinct lineages, with multiple lines of evidence showing strong reproductive isolation despite range overlap. We then scored thousands of citizen-science photos and identified four recognizable morphologies linked to these lineages. Within lineages, we detected regionally endemic subpopulations, connected by winds and currents, and identified individual long-distance dispersal events. We find that, even in these sailing species, genetic variation is highly partitioned geographically across the open ocean. Summary The open ocean is a vast and highly connected environment. The organisms that live there have a significant capacity for dispersal and few geographic boundaries to separate populations. Of these, the Portuguese man-o’-war or bluebottle (genus Physalia ) is uniquely suited to long-distance travel, using its gas-filled float and muscular crest to catch the wind and sail the sea surface. Physalia are distributed across the globe, and like many pelagic organisms, have been hypothesized to represent a massive, well-mixed population that extends across ocean basins. We tested this hypothesis by sequencing whole genomes of 133 samples collected from waters of over a dozen countries around the globe. Our results revealed five distinct lineages, with multiple lines of evidence indicating strong reproductive isolation, despite regions of range overlap. We combined these data with an independent dataset of thousands of images of Physalia uploaded to the citizen-science website inaturalist.org, which we scored for morphological characters including sail size, tentacle arrangement, and color. From these images, we identified four recognizable morphologies, described their geographical distribution, and linked them to four of the lineages identified with genomic data. We conclude there are at least four species, three of which correspond to species proposed by scientists in the 18th and 19th centuries: P. physalis , P utriculus , and P. megalista , along with one as yet unnamed species Physalia sp. from the Tasman Sea. Within each species, we observe significant population structure, with evidence of persistent subpopulations at a regional scale, as well as evidence for individual long-distance dispersal events. Our findings indicate that, instead of one well-mixed, cosmopolitan species, there are in fact multiple Physalia species with distinct but overlapping ranges, each made up of regionally endemic subpopulations that are connected by major ocean currents and wind patterns. ### Competing Interest Statement The authors have declared no competing interest.

The submediterranean is an ecotone distributed across southern Europe and north Africa, marking the transition between temperate and mediterranean climates. This is a remarkable climatic and vegetational dynamic area, with major shifts driven by changes in climate since the Late Quaternary. We explore future range shifts of the submediterranean ecotone caused by climate change using marcescent oaks as a proxy, considering their high prevalence in forests under such conditions. Our results confirm the ongoing dynamism of this ecotone, which is likely to expand its range northwards, where marcescent oaks will overlap Eurosiberian forests dominated by deciduous and temperate species. Conversely, in the southwestern Mediterranean, a loss of suitability is predicted, with remaining suitable habitat only at higher altitudes or along coastal regions, promoting scattered refuge suitable areas. To preserve these forests, further research is needed to assess the future ranges of individual species based on high-resolution models, to plan management and restoration strategies within a context of high disturbance by land use.

Canine lymphoma is one of the most common and aggressive hematopoietic tumors in dogs. Despite recent advances in veterinary cancer treatments, the lack of specificity, side effects, and resistance to conventional chemotherapies has opened an urgent need to develop more targeted and safe therapeutics to address this unmet need in dogs. Thus, in the present study, we aimed to generate a new class of therapeutics based on a recombinant single-domain antibody (sdAb) immunotoxin derived from the PE38 Pseudomonas aeruginosa exotoxin A. For this purpose, we fused the PE38 toxin with the specific C5 sdAb antibody, previously developed by our group for canine B-cell lymphoma. This resulted in a stable and highly specific C5-PE38 immunotoxin against canine B-cell lymphoma. The C5-PE38 immunotoxin revealed a potent cytotoxic activity (EC50 = 9.50 ± 0.04 μg/mL) against CLBL-1 canine B-cell lymphoma cells, while promoting inhibition of protein synthesis and, consequently, cell death. Importantly, in vivo results in a CLBL-1 xenograft mouse model demonstrated specific targeted tumor uptake and strong tumor growth inhibition in C5-PE38 treated mice compared with control vehicle-treated mice. The results obtained provide new data validating immunotoxins and recombinant sdAb-PE38 based scaffolds as a novel and promising anti-cancer therapy for the treatment of dog-related tumors, while contributing to comparative oncology.

Climate change poses a major threat to forests, impacting the distribution and viability of key species. Quercus pyrenaica Willd., a marcescent oak endemic to the Iberian Peninsula (Portugal and Spain) and southwestern France and a structural species in submediterranean forests, is particularly susceptible to shifts in temperature and precipitation patterns. Aiming to assess its potential loss of suitable area under future climate scenarios, we developed high-resolution spatial distribution models to project the future habitat suitability of Q. pyrenaica under two climate change scenarios (SSP3-7.0 and SSP5-8.5) for the periods 2070 and 2100. Our model, which has an excellent predictive performance (AUC of 0.971 and a TSS of 0.834), indicates a predominantly northward shift in the potential distribution of the species, accompanied by substantial habitat loss in southern and lowland regions. Long-term potential suitable area may shrink to 42% of that currently available. This, combined with the limited natural dispersal capacity of the species, highlights the urgency of targeted management and conservation strategies. These results offer critical insights to inform conservation strategies and forest management under ongoing climate change.

Persistent Memory (PM) enables the development of fast, persistent applications without employing costly HDD/SSD-based I/O operations. Since caches are volatile and CPUs may reorder and stall memory accesses for performance, developers must use low-level instructions to ensure a consistent state in case of a crash. Failure to do so can result in data corruption, data loss, or undefined behavior. In concurrent executions, this exposes a new class of bugs. HawkSet is an automatic, application-agnostic, and efficient tool to detect concurrent PM bugs. HawkSet uses lockset analysis, and automatic binary instrumentation to find all the bugs detected by the state-of-the-art tools and 7 previously unknown bugs. This is achieved without requiring application-specific knowledge or models, nor specialized debugging artifacts or guided executions. Compared to the state-of-the-art, HawkSet offers up to a 159x speedup, and consistently detects harder-to-reach bugs, where a rare interleaving is required.