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In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody–antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5′ and 3′ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo . A method to identify pairs of ligands that simultaneously bind to a target protein has been developed. The method uses two DNA-encoded chemical sub-libraries that self-assemble to form stable dual-display structures, and an encoding system that can be decoded by DNA sequencing and enables both ligands to be identified.

A sensitive, simple and reproducible protocol for nanoparticle-assisted laser desorption/ionization mass spectrometry imaging technique is described. The use of commercially available TiO2 nanoparticles abolishes heterogeneous crystallization, matrix background interferences and enhances signal detection, especially in the low mass range. Molecular image normalization was based on internal standard deposition on tissues, allowing direct comparison of drug penetration and distribution between different organs and tissues. The method was applied to analyze the distribution of the anticancer drug paclitaxel, inside normal and neoplastic mouse tissue sections. Spatial resolution was good, with a linear response between different in vivo treatments and molecular imaging intensity using therapeutic drug doses. This technique distinguishes the different intensity of paclitaxel distribution in control organs of mice, such as liver and kidney, in relation to the dose. Animals treated with 30 mg/kg of paclitaxel had half of the concentration of those treated with 60 mg/kg. We investigated the spatial distribution of paclitaxel in human melanoma mouse xenografts, following different dosage schedules and found a more homogeneous drug distribution in tumors of mice given repeated doses (5×8 mg/kg) plus a 60 mg/kg dose than in those assigned only a single 60 mg/kg dose. The protocol can be readily applied to investigate anticancer drug distribution in neoplastic lesions and to develop strategies to optimize and enhance drug penetration through different tumor tissues.

Projected climate changes in the Mediterranean exceed those in most European regions, yet their effects on vegetation remain uncertain. We investigated vegetation changes in the Agri Valley (Basilicata, Italy) using 318 plots, including 40 resurveys. Community-weighted Ellenberg indicator values (EIVs) and plant ecological groups were combined with long-term hydroclimatic anomalies reconstructed via the BIGBANG model (1951–2024), providing a long-term climatic baseline for interpretation. Significant shifts emerged in several EIVs, with clear habitat-specific patterns. Forests showed decreasing light and increasing moisture values, reflecting a higher presence of forest-associated species, though some diagnostic taxa declined. Grasslands exhibited increasing aridity, with a growing contribution of dry-grassland species and a decline in winter therophytes. Climatic analyses revealed pronounced long-term warming, accelerating after the 1980s, while annual precipitation remained highly variable without a monotonic trend. Recent years were marked by intensified drought, evidenced by declining SPEI values (2013–2022) and a higher frequency of dry months (SPEI ≤ −1). The convergence of vegetation responses, species turnover, and climatic anomalies supports climate-driven community trajectories. Despite limited land-use data, this multi-indicator framework effectively detects early ecological responses and identifies vulnerable habitats, providing valuable insights for the conservation and management of Mediterranean mountain ecosystems under ongoing climate change.

Antibody-cytokine fusion proteins (“immunocytokines”) represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8–IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8–IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4 + T cells 24 h after the administration of the combination. The fusion proteins F8–IL2 and L19–IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.

The combination of tumor-targeting IL2- and TNF-based antibody-cytokine fusions has exhibited encouraging results in mouse and men. Here, we studied their combination to assess efficacy and mechanism of action in four different immunocompetent mouse models of cancer. Mice receiving a single intratumoral injection of F8-IL2, F8-TNF or the combination were investigated for tumor-infiltrating leukocytes and rechallenged when cured. In three models, a proportion of treated animals could be cured, most probably by infiltrating NK and CD8 T cells. Most of the cured mice did not acquire protective immunity when rechallenged with the same tumor cell line. Immunocompetent mouse tumor models may not be adequate enough to predict the search for more efficacious therapy regimens. A single intralesional injection of two tumor-targeting antibody-cytokine fusion proteins led to tumor eradication in three out of four different tumor models. However, cured mice did not develop protective immunity upon tumor rechallenge.

The combined pressures of anthropogenic activities require rapid assessments of environmental impacts on ecosystems to develop strategies for sustainable management and biodiversity conservation. This study investigates the consequences of the anthropogenic effects in the upper Val d’Agri (Basilicata, Italy) by employing a multi-taxonomic analysis to investigate spatial dynamics and species responses to these pressures. By cataloguing 151 diverse species of small mammals, reptiles, birds, ground beetles, and lichens, we established a valuable baseline for analysing the local biodiversity. We assessed the relationship between anthropic impacts (industrial areas, roads, agriculture, forestry, etc.), vegetation cover, and species diversity. Our findings revealed a negative relationship between the number of species and proximity to industrial areas, emphasising the impact of these activities. Moreover, we observed a predominance of generalist biological traits across taxa in all the study sites rather than an increase in species specialisation with increased distance. This may suggest a homogenising effect caused by various anthropogenic activities that cause habitat fragmentation and species mortality, underlining the lasting influence of these activities on the biodiversity of the upper Agri Valley. Additionally, our study identifies numerous protected species, highlighting their vulnerability and emphasising the necessity for targeted conservation efforts to safeguard their existence.

Summary Phosphatidylserine (PS) and other anionic phospholipids, which become exposed on the surface of proliferating endothelial cells, tumor cells and certain leukocytes, have been used as targets for the development of clinical-stage biopharmaceuticals. One of these products (bavituximab) is currently being investigated in Phase 3 clinical trials. There are conflicting reports on the ability of bavituximab and other antibodies to recognize PS directly or through beta-2 glycoprotein 1, a serum protein that is not highly conserved across species. Here, we report on the generation and characterization of two fully human antibodies directed against phosphatidylserine. One of these antibodies (PS72) bound specifically to phosphatidylserine and to phosphatidic acid, but did not recognize other closely related phospholipids, while the other antibody (PS41) also bound to cardiolipin. Both PS72 and PS41 stained 8/9 experimental tumor models in vitro, but both antibodies failed to exhibit a preferential tumor accumulation in vivo, as revealed by quantitative biodistribution analysis. Our findings indicate that anionic phospholipids are exposed and accessible in most tumor types, but cast doubts about the possibility of efficiently targeting tumors in vivo with PS-specific reagents.

Questions: What are the main drivers of variation in beta-diversity for Bromus erectus semi-natural dry grasslands of habitat 6210(*) at different scales? How should environmental variables and spatial patterns be taken into account to conserve the maximum possible beta-diversity within the habitat? Location: Central Italy. Methods: We used 195 vegetation relevés distributed in three nested extents: a single mountain, a mountain chain and southern Lazio. Multiple regression on distance matrices was performed using dissimilarity matrices based on: (1) species abundances as response variables; (2) spatial coordinates and environmental parameters (altitude, slope, percentage of rock and stone coverage, aspect, annual rainfall) as explanatory variables. The two groups of explanatory variables were used separately to partition the variation, and jointly to assess the relative contribution of each individual variable. Those variables found to significantly affect beta-diversity were used to: (1) compare beta-diversity levels between a set of randomly selected and a set of stratified relevés; and (2) analyse the habitat distribution across environmental gradients. These analyses, together with the curves describing the relationships between spatial distances and composition dissimilarities, were used to inform management decisions for the habitat. Results: Most of the variance was explained by environmental variables, whose share was higher in the smallest and intermediate extent than in the broadest extent. Community dissimilarity increased in proportion to differences in altitude and spatial distances at every extent. Accordingly, at all the extents, the selection of relevés stratified by altitude or selected taking into account a minimum spatial distance included significantly higher levels of within-habitat betadiversity, than randomly selected relevés. The relation of beta-diversity to the variation in aspect and annual rainfall varied at different extents. Conclusions: Our results demonstrate that dry grassland management plans aimed at conserving the maximum within-habitat beta-diversity should take into account variation in environmental variables, among which altitude proved to be a critical factor at every extent. Also, spatial distances positively affect within-habitat beta-diversity levels, and scale-dependent minimum distances among habitat patches should be taken into account when selecting patches of habitat 6210(*) to be conserved in the study area.

Questions What are the main drivers of variation in beta-diversity for B romus erectus semi-natural dry grasslands of habitat 6210(*) at different scales? How should environmental variables and spatial patterns be taken into account to conserve the maximum possible beta-diversity within the habitat? Location Central I taly. Methods We used 195 vegetation releves distributed in three nested extents: a single mountain, a mountain chain and southern L azio. Multiple regression on distance matrices was performed using dissimilarity matrices based on: (1) species abundances as response variables; (2) spatial coordinates and environmental parameters (altitude, slope, percentage of rock and stone coverage, aspect, annual rainfall) as explanatory variables. The two groups of explanatory variables were used separately to partition the variation, and jointly to assess the relative contribution of each individual variable. Those variables found to significantly affect beta-diversity were used to: (1) compare beta-diversity levels between a set of randomly selected and a set of stratified releves; and (2) analyse the habitat distribution across environmental gradients. These analyses, together with the curves describing the relationships between spatial distances and composition dissimilarities, were used to inform management decisions for the habitat. Results Most of the variance was explained by environmental variables, whose share was higher in the smallest and intermediate extent than in the broadest extent. Community dissimilarity increased in proportion to differences in altitude and spatial distances at every extent. Accordingly, at all the extents, the selection of releves stratified by altitude or selected taking into account a minimum spatial distance included significantly higher levels of within-habitat beta-diversity, than randomly selected releves. The relation of beta-diversity to the variation in aspect and annual rainfall varied at different extents. Conclusions Our results demonstrate that dry grassland management plans aimed at conserving the maximum within-habitat beta-diversity should take into account variation in environmental variables, among which altitude proved to be a critical factor at every extent. Also, spatial distances positively affect within-habitat beta-diversity levels, and scale-dependent minimum distances among habitat patches should be taken into account when selecting patches of habitat 6210(*) to be conserved in the study area. We identified the drivers of variation in dry grasslands [beta]-diversity at multiple scales to help managers define conservation priorities. Our results demonstrate that environmental variables, especially altitude, are critical factors affecting the within-habitat [beta]-diversity levels. Moreover scale-dependent minimum distances among habitat patches should be taken into account when selecting grasslands patches to be conserved.