Meta-analysis of livestock effects on tree regeneration in oak agroforestry systems.

Livestock grazing occupies over a quarter of terrestrial land and is prevalent to agroforestry ecosystems, potentially affecting the survival, growth, and density of trees' early developmental stages, such as seeds, seedlings, and saplings. To address the effects of livestock on tree recruitment in the face of ongoing debates about their impacts, we conducted a 33-year meta-analysis in Quercus-dominated agroforestry systems. Our analysis revealed a consistently negative effect of livestock on oak acorns, seedlings, and saplings. Significantly, livestock body size influenced oak regeneration, with small-sized livestock, notably sheep and goats, having a more pronounced negative impact compared to mixed-size systems, mainly involving cattle and sheep. The effects of small-sized livestock were markedly detrimental on acorn survival and seedling/sapling density, although no studies eligible for meta-analysis examined large livestock impacts on acorns. Overall, mixed-size livestock systems, often involving cattle and sheep, lessen the negative effects. Our findings indicate that the body size and foraging behaviors of livestock should be considered for the ecological sustainability of the tree component in agroforestry systems. While protective measures have long been integral to well-managed agroforestry systems, our results underscore the importance of integrating diverse livestock sizes and applying specific protective strategies, particularly for acorns and saplings, to further refine these practices. Future research should expand to underrepresented regions and livestock types to refine global agroforestry management practices.

Comprehensive clinical imaging, histopathological analysis and liquid biopsy-based surveillance of human uveal melanoma in a prolonged rabbit xenograft model.

Uveal melanoma is the most common intraocular tumor in adults. Our group has previously developed a human uveal melanoma animal model; however, adverse effects caused by the immunosuppressive agent, cyclosporine A, prevented animals from surviving more than 12 weeks. In this study, we tested multiple cyclosporine A doses over an extended disease course up to 20 weeks, providing complete clinical imaging of intraocular tumors, histopathological analysis and liquid biopsy biomarker analysis. Twenty albino rabbits were divided into four groups with different daily cyclosporine A schedules (0-10 mg/kg) and inoculated with human uveal melanoma cell lines, 92.1 or MP41, into the suprachoroidal space. Rabbits were monitored with fundoscopy, ultrasound and optical coherence tomography. Intraocular tumors (macroscopic or microscopic) were detected in all study animals. Tumor size and growth were correlated to cyclosporine A dose, with tumors regressing when cyclosporine A was arrested. All tumors expressed HMB-45 and MelanA; however, tumor size, pigmentation and cell morphology differed in 92.1 vs. MP41 tumors. Finally, across all groups, circulating tumor DNA from plasma and aqueous humor was detected earlier than tumor detection by imaging and correlated to tumor growth. In conclusion, using three clinically relevant imaging modalities (fundoscopy, ultrasonography and optical coherence tomography) and liquid biopsy, we were successfully able to monitor tumor progression in our rabbit xenograft model of human uveal melanoma.

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions.

BACKGROUND: Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS: Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS: UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS: Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Ungulates mitigate the effects of drought and shrub encroachment on the fire hazard of Mediterranean oak woodlands.

Climate change is increasing the frequency of droughts and the risk of severe wildfires, which can interact with shrub encroachment and browsing by wild ungulates. Wild ungulate populations are expanding due, among other factors, to favorable habitat changes resulting from land abandonment or land-use changes. Understanding how ungulate browsing interacts with drought to affect woody plant mortality, plant flammability, and fire hazard is especially relevant in the context of climate change and increasing frequency of wildfires. The aim of this study is to explore the combined effects of cumulative drought, shrub encroachment, and ungulate browsing on the fire hazard of Mediterranean oak woodlands in Portugal. In a long-term (18 years) ungulate fencing exclusion experiment that simulated land abandonment and management neglect, we investigated the population dynamics of the native shrub Cistus ladanifer, which naturally dominates the understory of woodlands and is browsed by ungulates, comparing areas with (no fencing) and without (fencing) wild ungulate browsing. We also modeled fire behavior in browsed and unbrowsed plots considering drought and nondrought scenarios. Specifically, we estimated C. ladanifer population density, biomass, and fuel load characteristics, which were used to model fire behavior in drought and nondrought scenarios. Overall, drought increased the proportion of dead C. ladanifer shrub individuals, which was higher in the browsed plots. Drought decreased the ratio of live to dead shrub plant material, increased total fuel loading, shrub stand flammability, and the modeled fire parameters, that is, rate of surface fire spread, fireline intensity, and flame length. However, total fuel load and fire hazard were lower in browsed than unbrowsed plots, both in drought and nondrought scenarios. Browsing also decreased the population density of living shrubs, halting shrub encroachment. Our study provides long-term experimental evidence showing the role of wild ungulates in mitigating drought effects on fire hazard in shrub-encroached Mediterranean oak woodlands. Our results also emphasize that the long-term effects of land abandonment can interact with climate change drivers, affecting wildfire hazard. This is particularly relevant given the increasing incidence of land abandonment.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

A Comparison of Clear Cell Sarcoma to Jaw and Salivary Tumors Bearing EWS Fusions.

OBJECTIVE: To review tumors identified as "clear cell sarcoma" in order to determine similarities to the rare EWS fusion positive jaw and salivary gland tumors clear cell odontogenic carcinoma (CCOC) and clear cell carcinoma of the salivary gland (CCC). METHODS: PubMed was used to collect all reports of clear cell sarcoma (CCS). Search parameters were "clear cell sarcoma" and "CCS." References in the publications were screened and cross-referenced. Data extracted included demographic characteristics, presenting signs and symptoms, radiographic findings, histological and immunohistochemical features and known molecular/genetic aberrations. RESULTS: Clear cell sarcoma has several similarities to CCOC and CCC. All three tumor types have similar histologic appearances including the presence of clear cells, as well as similar genetic profiles in that all harbor an EWSR1-CREB family fusions. Additionally, these tumors appear in soft tissue as well as bone, and can have a prolonged clinical course. CCS can appear anywhere in the body, including the head and neck region. All three tumors appear to have a predilection to women, although CCS may have a slight younger age of onset as compared to CCOC and CCC (3rd vs 5th decade of life, respectively). CONCLUSION: Gaining a better understanding of the similarities and differences between these three tumors may lead to a better understanding of each one.

CIC-DUX4 Chromatin Profiling Reveals New Epigenetic Dependencies and Actionable Therapeutic Targets in CIC-Rearranged Sarcomas.

CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.

DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins.

Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.

Drivers of soil microbial and detritivore activity across global grasslands.

Covering approximately 40% of land surfaces, grasslands provide critical ecosystem services that rely on soil organisms. However, the global determinants of soil biodiversity and functioning remain underexplored. In this study, we investigate the drivers of soil microbial and detritivore activity in grasslands across a wide range of climatic conditions on five continents. We apply standardized treatments of nutrient addition and herbivore reduction, allowing us to disentangle the regional and local drivers of soil organism activity. We use structural equation modeling to assess the direct and indirect effects of local and regional drivers on soil biological activities. Microbial and detritivore activities are positively correlated across global grasslands. These correlations are shaped more by global climatic factors than by local treatments, with annual precipitation and soil water content explaining the majority of the variation. Nutrient addition tends to reduce microbial activity by enhancing plant growth, while herbivore reduction typically increases microbial and detritivore activity through increased soil moisture. Our findings emphasize soil moisture as a key driver of soil biological activity, highlighting the potential impacts of climate change, altered grazing pressure, and eutrophication on nutrient cycling and decomposition within grassland ecosystems.

Multidimensional responses of grassland stability to eutrophication.

Eutrophication usually impacts grassland biodiversity, community composition, and biomass production, but its impact on the stability of these community aspects is unclear. One challenge is that stability has many facets that can be tightly correlated (low dimensionality) or highly disparate (high dimensionality). Using standardized experiments in 55 grassland sites from a globally distributed experiment (NutNet), we quantify the effects of nutrient addition on five facets of stability (temporal invariability, resistance during dry and wet growing seasons, recovery after dry and wet growing seasons), measured on three community aspects (aboveground biomass, community composition, and species richness). Nutrient addition reduces the temporal invariability and resistance of species richness and community composition during dry and wet growing seasons, but does not affect those of biomass. Different stability measures are largely uncorrelated under both ambient and eutrophic conditions, indicating consistently high dimensionality. Harnessing the dimensionality of ecological stability provides insights for predicting grassland responses to global environmental change.

The positive effect of plant diversity on soil carbon depends on climate.

Little is currently known about how climate modulates the relationship between plant diversity and soil organic carbon and the mechanisms involved. Yet, this knowledge is of crucial importance in times of climate change and biodiversity loss. Here, we show that plant diversity is positively correlated with soil carbon content and soil carbon-to-nitrogen ratio across 84 grasslands on six continents that span wide climate gradients. The relationships between plant diversity and soil carbon as well as plant diversity and soil organic matter quality (carbon-to-nitrogen ratio) are particularly strong in warm and arid climates. While plant biomass is positively correlated with soil carbon, plant biomass is not significantly correlated with plant diversity. Our results indicate that plant diversity influences soil carbon storage not via the quantity of organic matter (plant biomass) inputs to soil, but through the quality of organic matter. The study implies that ecosystem management that restores plant diversity likely enhances soil carbon sequestration, particularly in warm and arid climates.

Non-canonical integrin signaling activates EGFR and RAS-MAPK-ERK signaling in small cell lung cancer.

Background: Small cell lung cancer (SCLC) is an extremely aggressive cancer type with a patient median survival of 6-12 months. Epidermal growth factor (EGF) signaling plays an important role in triggering SCLC. In addition, growth factor-dependent signals and alpha-, beta-integrin (ITGA, ITGB) heterodimer receptors functionally cooperate and integrate their signaling pathways. However, the precise role of integrins in EGF receptor (EGFR) activation in SCLC remains elusive. Methods: We analyzed human precision-cut lung slices (hPCLS), retrospectively collected human lung tissue samples and cell lines by classical methods of molecular biology and biochemistry. In addition, we performed RNA-sequencing-based transcriptomic analysis in human lung cancer cells and human lung tissue samples, as well as high-resolution mass spectrometric analysis of the protein cargo from extracellular vesicles (EVs) that were isolated from human lung cancer cells. Results: Our results demonstrate that non-canonical ITGB2 signaling activates EGFR and RAS/MAPK/ERK signaling in SCLC. Further, we identified a novel SCLC gene expression signature consisting of 93 transcripts that were induced by ITGB2, which may be used for stratification of SCLC patients and prognosis prediction of LC patients. We also found a cell-cell communication mechanism based on EVs containing ITGB2, which were secreted by SCLC cells and induced in control human lung tissue RAS/MAPK/ERK signaling and SCLC markers. Conclusions: We uncovered a mechanism of ITGB2-mediated EGFR activation in SCLC that explains EGFR-inhibitor resistance independently of EGFR mutations, suggesting the development of therapies targeting ITGB2 for patients with this extremely aggressive lung cancer type.

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Development of Cationic Lipid LAH4-L1 siRNA Complexes for Focused Ultrasound Enhanced Tumor Uptake.

RNAi has considerable potential as a cancer therapeutic approach, but effective and efficient delivery of short interfering RNA (siRNA) to tumors remains a major hurdle. It remains a challenge to prepare a functional siRNA complex, target enough dose to the tumor, and stimulate its internalization into tumor cells and its release to the cytoplasm. Here, we show how these key barriers to siRNA delivery can be overcome with a complex─comprising siRNA, cationic lipids, and pH-responsive peptides─that is suited to tumor uptake enhancement via focused ultrasound (FUS). The complex provides effective nucleic acid encapsulation, nuclease protection, and endosomal escape such that gene silencing in cells is substantially more effective than that obtained with either equivalent lipoplexes or commercial reagents. In mice bearing MDA-MB-231 breast cancer xenografts, both lipid and ternary, lipid:peptide:siRNA complexes, prepared with near-infrared fluorescently labeled siRNA, accumulate in tumors following FUS treatments. Therefore, combining a well-designed lipid:peptide:siRNA complex with FUS tumor treatments is a promising route to achieve robust in vivo gene delivery.

Clinical and anatomic correlates of objectively measured zonular dehiscence in 427 postmortem pseudophakic eyes.

OBJECTIVE: This study aims to objectively measure the degree of zonular dehiscence in postmortem eyes and to assess for clinical and anatomic correlates. DESIGN: Cross-sectional study. MATERIALS: Four hundred and twenty-seven postmortem pseudophakic human eyes. METHODS: Eyes were obtained from the Lions Gift of Sight Eye Bank. Microscope photographs were taken of the eyes in Miyake-Apple view, and region-of-interest analysis was performed using ImageJ to measure the area, circumference, and diameter of the capsular bag, the ciliary ring, and the capsulorhexis. Clinical and anatomic parameters were assessed using simple linear regression analysis and one-way analysis of variance with post hoc Bonferroni testing. Zonular dehiscence was measured via 2 surrogates: capsule area over ciliary ring area ratio (CCR) and capsule-ciliary ring decentration (CCD). Low CCR and high CCD indicate more zonular dehiscence. RESULTS: CCR was significantly inversely correlated with smaller capsulorhexi (p = 0.012), lower intraocular lens power (p < 0.00001), younger age at death (p = 0.00002), and a longer cataract-to-death time (p = 0.00786). CCR also was significantly lower in glaucomatous cases (p = 0.0291). CCD was significantly correlated with longer cataract-to-death time (p = 0.000864), larger ciliary ring area (p = 0.001), more posterior capsule opacification (p = 0.0234), and higher Soemmering's ring opacity (p = 0.0003). There was also significantly more decentration in male eyes than in female eyes (p = 0.00852). CONCLUSIONS: CCR and CCD are novel measures of zonular dehiscence in postmortem eyes, with many interesting correlates. An enlarged ciliary ring area is possibly associated with zonular dehiscence in pseudophakic eyes and may be a quantifiable surrogate in vivo.

The ETS transcription factor ETV6 constrains the transcriptional activity of EWS-FLI to promote Ewing sarcoma.

Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS-FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS-FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

Clinical Implications and Treatment Strategies for ESR1 Fusions in Hormone Receptor-Positive Metastatic Breast Cancer: A Case Series.

In hormone receptor-positive metastatic breast cancer (HR+ MBC), endocrine resistance is commonly due to genetic alterations of ESR1, the gene encoding estrogen receptor alpha (ERα). While ESR1 point mutations (ESR1-MUT) cause acquired resistance to aromatase inhibition (AI) through constitutive activation, far less is known about the molecular functions and clinical consequences of ESR1 fusions (ESR1-FUS). This case series discusses 4 patients with HR+ MBC with ESR1-FUS in the context of the existing ESR1-FUS literature. We consider therapeutic strategies and raise the hypothesis that CDK4/6 inhibition (CDK4/6i) may be effective against ESR1-FUS with functional ligand-binding domain swaps. These cases highlight the importance of screening for ESR1-FUS in patients with HR+ MBC while continuing investigation of precision treatments for these genomic rearrangements.

Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture.

In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorized as therapeutic against bacterial infections. The use of antimicrobials in aquaculture may involve a broad environmental application that affects a wide variety of bacteria, promoting the spread of bacterial resistance genes. Probiotics and bacteriocins, antimicrobial peptides produced by some types of lactic acid bacteria (LAB), have been successfully tested in aquatic animals as alternatives to control bacterial infections. Supplementation might have beneficial impacts on the intestinal microbiota, immune response, development, and/or weight gain, without the issues associated with antibiotic use. Thus, probiotics and bacteriocins represent feasible alternatives to antibiotics. Here, we provide an update with respect to the relevance of aquaculture in the animal protein production sector, as well as the present and future challenges generated by outbreaks and antimicrobial resistance, while highlighting the potential role of probiotics and bacteriocins to address these challenges. In addition, we conducted data analysis using a simple linear regression model to determine whether a linear relationship exists between probiotic dose added to feed and three variables of interest selected, including specific growth rate, feed conversion ratio, and lysozyme activity.

Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.