Microenvironmental Conditions Research Articles

Rapid Analysis of Soil Organic Carbon in Agricultural Lands: Potential of Integrated Image Processing and Infrared Spectroscopy

The significance of soil in the agricultural industry is profound, with healthy soil representing an important role in ensuring food security. In addition, soil is the largest terrestrial carbon sink on earth. The soil carbon pool is composed of both inorganic and organic forms. The equilibrium of the soil carbon pool directly impacts the carbon cycle via all of the other processes on the planet. With the development of agricultural systems from traditional to conventional ones, and with the current era of precision agriculture, which involves making decisions based on information, the importance of understanding soil is becoming increasingly clear. The control of microenvironment conditions and soil fertility represents a key factor in achieving higher productivity in these systems. Furthermore, agriculture represents a significant contributor to carbon emissions, a topic that has become timely given the necessity for carbon neutrality. In addition to these concerns, updating soil-related data, including information on macro and micronutrient conditions, is important. Carbon represents one of the major nutrients for crops and plays a key role in the retention and release of other nutrients and the management of soil physical properties. Despite the importance of carbon, existing analytical methods are complex and expensive. This discourages frequent analyses, which results in a lack of soil carbon-related data for agricultural fields. From this perspective, in situ soil organic carbon (SOC) analysis can provide timely management information for calibrating fertilizer applications based on the soil–carbon relationship to increase soil productivity. In addition, the available data need frequent updates due to rapid changes in ecosystem services and the use of extensive fertilizers and pesticides. Despite the importance of this topic, few studies have investigated the potential of image analysis based on image processing and spectral data recording. The use of spectroscopy and visual color matching to develop SOC predictions has been considered, and the use of spectroscopic instruments has led to increased precision. Our extensive literature review shows that color models, especially Munsell color charts, are better for qualitative purposes and that Cartesian-type color models are appropriate for quantification. Even for the color model, spectroscopy data could be used, and these data have the potential to improve the precision of measurements. On the other hand, mid-infrared radiation (MIR) and near-infrared radiation (NIR) diffuse reflection has been reported to have a greater ability to predict SOC. Finally, this article reports the availability of inexpensive portable instruments that can enable the development of in situ SOC analysis from reflection and emission information with the integration of images and spectroscopy. This integration refers to machine learning algorithms with a reflection-oriented spectrophotometer and emission-based thermal images which have the potential to predict SOC without the need for expensive instruments and are easy to use in farm applications.

Influence of construction materials on the natural mummification process: case study of the municipal pantheon of Santa Paula, Guanajuato, Mexico.

The process of natural or spontaneous mummification has been observed for more than a hundred years in various Mexican pantheons. The construction materials used in the niches of the pantheons generate particular microenvironmental conditions that promote the preservation of organic materials. The construction materials (mining adobe, brick and stone material) that make up the three series of the municipal pantheon of Santa Paula were studied and analyzed using X-ray Diffraction, X-ray Fluorescence and petrographic analysis techniques. At the same time, the mechanical and physical properties of the materials were determined, as well as the relative humidity and temperature within the niches. The results indicate that the geochemistry, distribution and percentage of said materials inside of the niches created exceptional microclimatic conditions that favored the conservation of the bodies. With this contribution we intend to advance the understanding of the influence that construction materials had on the process of natural mummification.

Radial Oxygen Loss Triggers Diel Fluctuation of Cadmium Dissolution in the Rhizosphere of Rice.

Cadmium (Cd) contamination poses a significant global threat to human health, primarily through dietary intake, with rice serving as a major source. While Cd predominantly resides in bound states in soil, the physiological processes by which rice facilitates Cd absorption in the rhizosphere remain largely elusive. This study delves into the mechanisms governing Cd uptake by rice plants in the rhizosphere, emphasizing the impact of daytime and nighttime fluctuations in microenvironmental conditions. Employing a microfluidic chip setup, the research reveals that radial oxygen loss from rice roots triggers dissolution of Cd in the rhizosphere. Notably, Cd mobility exhibits distinct diurnal fluctuations, peaking at 44.0 ± 4.1 nM during the daytime and dropping to 8.3 ± 1.3 nM during the nighttime. Further investigations reveal that variations in dissolved oxygen and hydroxyl radical concentrations influence Cd release, while pH changes and microbial reduction reactions play crucial roles in Cd immobilization. These findings provide insights into the intricate processes governing Cd mobilization in the rice rhizosphere, highlighting the importance of regulating these processes for effective Cd adsorption control in rice crops and safeguarding public health.

Preparation of Biomimetic Selenium-Baicalein Nanoparticles and Their Targeted Therapeutic Application in Nonsmall Cell Lung Cancer.

In this study, we prepared bionic selenium-baicalein nanoparticles (ACM-SSe-BE) for the targeted treatment of nonsmall cell lung cancer. Due to the coating of the A549 membrane, the system has homologous targeting capabilities, allowing for the preparation of target tumor cells. The borate ester bond between selenium nanoparticles (SSe) and baicalein (BE) is pH-sensitive and can break under acidic conditions in the tumor microenvironment to achieve the targeted release of BE at the tumor site. Moreover, SSe further enhances the antitumor effect of BE by increasing the production of ROS in tumor cells. Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) showed that the ACM-SSe-BE had a particle size of approximately 155 ± 2 nm. FTIR verified the successful coupling of SSe and BE. In vitro release experiments indicated that the cumulative release of ACM-SSe-BE at pH 5.5 after 24 h was 69.39 ± 1.07%, which was less than the 20% release at pH 7.4, confirming the pH-sensitive release of BE in ACM-SSe-BE. Cell uptake experiments and in vivo imaging showed that ACM-SSe-BE had good targeting ability. The results of MTT, flow cytometry, Western blot, and cell immunofluorescence staining demonstrated that ACM-SSe-BE promoted A549 cell apoptosis and inhibited cell proliferation. The in vivo antitumor results were consistent with those of the cell experiments. These results clearly suggested that ACM-SSe-BE will be a promising bionic nanosystem for the treatment of nonsmall cell lung cancer.

Ultrasound-Activated Probiotics Vesicles Coating for Titanium Implant Infections Through Bacterial Cuproptosis-Like Death and Immunoregulation.

Implant-associated infections (IAIs) are the main cause of prosthetic implant failure. Bacterial biofilms prevent antibiotic penetration, and the unique metabolic conditions in hypoxic biofilm microenvironment may limit the efficacy of conventional antibiotic treatment. Escaping survival bacteria may not be continually eradicated, resulting in the recurrence of IAIs. Herein, a sonosensitive metal-organic framework of Cu-TCPP (tetrakis(4-carboxyphenyl) porphyrin) nanosheets and tinidazole doped probiotic-derived membrane vesicles (OMVs) with high-penetration sonodynamic therapy (SDT), bacterial metabolic state interference, and bacterial cuproptosis-like death to eradicate IAIs is proposed. The Cu-TCPP can convert O2 to toxic 1O2 through SDT in the normoxic conditions, enhancing the hypoxic microenvironment and activating the antibacterial activity of tinidazole. The released Cu(II) under ultrasound can be converted to Cu(I) by exogenous poly(tannic acid) (pTA) and endogenous glutathione. The disruption of the bacterial membrane by SDT can enhance the Cu(I) transporter activity. Transcriptomics indicate that the SDT-enhanced Cu(I) overload and hypoxia-activated therapy hinder the tricarboxylic acid cycle (TCA), leading to bacterial cuproptosis-like death. Moreover, the OMVs-activated therapy can polarize macrophages to a M2-like phenotype and facilitate bone repair. The sonodynamic biofilm microenvironment modulation strategy, whereby the hypoxia-enhanced microenvironment is potentiated to synergize SDT with OMVs-activated therapy, provides an effective strategy for antibacterial and osteogenesis performance.

Hypoxia-Driven Changes in Tumor Microenvironment: Insights into Exosome-Mediated Cell Interactions.

Hypoxia, as a prominent feature of the tumor microenvironment, has a profound impact on the multicomponent changes within this environment. Under hypoxic conditions, the malignant phenotype of tumor cells, the variety of cell types within the tumor microenvironment, as well as intercellular communication and material exchange, undergo complex alterations. These changes provide significant prospects for exploring the mechanisms of tumor development under different microenvironmental conditions and for devising therapeutic strategies. Exosomes secreted by tumor cells and stromal cells are integral components of the tumor microenvironment, serving as crucial mediators of intercellular communication and material exchange, and have consequently garnered increasing attention from researchers. This review focuses on the mechanisms by which hypoxic conditions promote the release of exosomes by tumor cells and alter their encapsulated contents. It also examines the effects of exosomes derived from tumor cells, immune cells, and other cell types under hypoxic conditions on the tumor microenvironment. Additionally, we summarize current research progress on the potential clinical applications of exosomes under hypoxic conditions and propose future research directions in this field.

Pharmacologic Inhibition of EIF4A Blocks NRF2 Synthesis to Prevent Osteosarcoma Metastasis.

Effective therapies for metastatic osteosarcoma (OS) remain a critical unmet need. Targeting mRNA translation in metastatic OS offers a promising option, as selective translation drives the synthesis of cytoprotective proteins under harsh microenvironmental conditions to facilitate metastatic competence. We assessed the expression levels of eukaryotic translation factors in OS, revealing the high expression of the eukaryotic initiation factor 4A1 (EIF4A1). Using a panel of metastatic OS cell lines and patient-derived xenograft (PDX) models, EIF4A1 inhibitors were evaluated for their ability to block proliferation and reduce survival under oxidative stress, mimicking harsh conditions of the lung microenvironment. Inhibitors were also evaluated for their antimetastatic activity using the ex vivo pulmonary metastasis assay and in vivo metastasis models. Proteomics was performed to catalog which cytoprotective proteins or pathways were affected by EIF4A1 inhibition. CR-1-31B, a rocaglate-based EIF4A1 inhibitor, exhibited nanomolar cytotoxicity against all metastatic OS models tested. CR-1-31B exacerbated oxidative stress and apoptosis when OS cells were co-treated with tert-butylhydroquinone, a chemical oxidative stress inducer. CR-1-31B potently inhibited OS growth in the pulmonary metastasis assay model and in experimental and spontaneous models of OS lung metastasis. Proteomic analysis revealed that tert-butylhydroquinone-mediated upregulation of the NRF2 antioxidant factor was blocked by co-treatment with CR-1-31B. Genetic inactivation of NRF2 phenocopied the antimetastatic activity of CR-1-31B. Finally, the clinical-grade EIF4A1 phase-1-to-2 inhibitor, zotatifin, similarly blocked NRF2 synthesis and the OS metastatic phenotype. Collectively, our data reveal that pharmacologic targeting of EIF4A1 is highly effective in blocking OS metastasis by blunting the NRF2 antioxidant response.

Estimating Impacts of p16 Status on Tumor Radiosensitivity in Head and Neck Cancer using Predictive Models.

The intrinsic radiosensitivity index (RSI) and genomic-adjusted radiation dose (GARD) were reported to be able to predict the surviving fraction at 2 Gy and therapeutic effect when delivering actual treatment doses using the gene expression profiles of clinical cases. Given the impact of p16 status, a surrogate marker of the human papillomavirus (HPV) infection, on radiosensitivity, we attempted to apply the RSI and GARD to estimate p16-associated radiosensitivity in head and neck squamous cell carcinoma (HNSC). For this purpose, The Cancer Genome Atlas (TCGA) dataset was employed. In the GARD calculation, we assumed that p16-positive patients received 60 Gy in 30 fractions, while p16-negative patients received 70 Gy in 35 fractions. p16 positivity was associated with favorable characteristics compared to negative patients. The RSI and GARD analyses demonstrated increased radiosensitivity and high therapeutic effect in p16-positive patients, compared to p16-negative patients. Additionally, tumor microenvironmental conditions predicted by other models were also significantly affected by p16 status. Collectively, the models used in this study could be a promising tool for estimating p16-associated radiosensitivity in HNSC.

Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5β1 integrins

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood.Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5β1 integrins are involved.In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detectingtools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.

Stimuli Responsive Molecular Exchange of Structure Directing Agents on Gold Nanobipyramids: Cancer Cell Detection and Synergistic Therapeutics.

Surface-engineered gold nanoparticles have been considered as versatile systems for theranostics applications. Moreover, surface covering or stabilizing agents on gold nanoparticles especially gold nanobipyramids (AuNBPs) provides an extra space for cargo molecules entrapment. However, it is not well studied yet and also the preparation of AuNBPs still remains dependent largely on cetyltrimethylammonium bromide (CTAB), a cytotoxic surfactant. Therefore, the direct use of CTAB stabilized nanoparticles is not recommended for cancer theranostics applications. Herein, we address an approach of dodecyl ethyl dimethylammonium bromide (DMAB) as biocompatible structure directing agent for AuNBPs, which also accommodate anticancer drug doxorubicin (45%), an additional chemotherapeutics agent. Upon near-infrared light (NIR, 808 nm) exposure, engineered AuNBPs exhibit (i) better phototransduction (51 °C) due to NIR absorption ability (650-900 nm), (ii) photo triggered drug release (more than 80%), and (iii) synergistic chemophototherapy for breast cancer cells. Drug release response has been evaluated in tumor microenvironment conditions (84% in acidic pH and 80% at high GSH) due to protonation and high affinity of thiol binding with AuNBPs followed by DMAB replacement. Intracellular glutathione (GSH, 5-7.5 mM) replaces DMAB from AuNBPs, which cause easy aggregation of nanoparticles as corroborated by colorimetric shifts, suggesting their utilization as a molecular sensing probe of early stage cancer biomarkers. Our optimized recipe yield is monodisperse DMAB-AuNBPs with ∼90% purity even at large scales (500 mL volume per batch). DMAB-AuNBPs show better cell viability (more than 90%) across all concentrations (5-500 ug/mL) when directly compared to CTAB-AuNBPs (less than 10%). Our findings show the potential of DMAB-AuNBPs for early stage cancer detection and theranostics applications.

Mitochondrial Proteins as Metabolic Biomarkers and Sites for Therapeutic Intervention in Primary and Metastatic Cancers.

Accelerated aerobic glycolysis is one of the main metabolic alterations in cancer, associated with malignancy and tumor growth. Although glycolysis is one of the most studied properties of tumor cells, recent studies demonstrate that oxidative phosphorylation (OxPhos) is the main ATP provider for the growth and development of cancer. In this last regard, the levels of mRNA and protein of OxPhos enzymes and transporters (including glutaminolysis, acetate and ketone bodies catabolism, free fatty acid β-oxidation, Krebs Cycle, respiratory chain, phosphorylating system- ATP synthase, ATP/ADP translocator, Pi carrier) are altered in tumors and cancer cells in comparison to healthy tissues and organs, and non-cancer cells. Both energy metabolism pathways are tightly regulated by transcriptional factors, oncogenes, and tumor-suppressor genes, all of which dictate their protein levels depending on the micro-environmental conditions and the type of cancer cell, favoring cancer cell adaptation and growth. In the present review paper, variation in the mRNA and protein levels as well as in the enzyme/ transporter activities of the OxPhos machinery is analyzed. An integral omics approach to mitochondrial energy metabolism pathways may allow for identifying their use as suitable, reliable biomarkers for early detection of cancer development and metastasis, and for envisioned novel, alternative therapies.

Unraveling local and regional determinants of high plant diversity at marine rocky outcrops in Uruguay

AbstractQuestionsMarine rocky outcrops are model systems of community assembly under harsh conditions. The prevailing environmental conditions that determine a coast–inland gradient of stress and disturbance impose a strong environmental filter on community assembly. However, the Atlantic rocky outcrops of Uruguay challenge this assumption as they exhibit high plant diversity at small spatial scales. We investigated this study system to answer: (1) which environmental factors determine diversity at the local scale; (2) and how does the regional pool influence species composition along the coast–inland gradient?MethodsWe analyzed the local and regional determinants of plant community assembly in four rocky sites along the Uruguayan coast. Through a random sampling approach stratified by distance to the coast, we recorded species occurrences and microenvironmental conditions. Observed taxa were assigned into different species assemblages according to their typical vegetation type. Generalized Linear Model (GLM)–Poisson regression, quantile regressions and hierarchical cluster analyses were used to identify the factors underlying local richness and the spatial vegetation structure.ResultsNine different vegetation types generate a mass effect that enhances local diversity. Unexpectedly, 56% of the species are from non‐marine environments — e.g. grasslands or hydrophilous herblands. Species assemblages were spatially clustered, with a turnover of these clusters along the coast–inland gradient. Elevation, distance to the coast and vegetation cover promoted richness, likely due to an attenuation of marine filters. Meanwhile, substrate availability reduced richness, likely due to competitive exclusion as a result of more resources and homogeneous microenvironmental conditions.ConclusionsThe high local diversity along the marine rocky outcrops of Uruguay is best explained by a functionally diverse regional species pool, which interacts with local heterogeneity, and by conditions that enhance or attenuate the effect of marine stress. Effective conservation and management strategies are key to minimize the detrimental effects of urbanization, fragmentation, and non‐native plant invasions on these diverse habitats.

Computational model of the cancer necrotic core formation in a tumor-on-a-chip device

The mechanisms underlying the formation of necrotic regions within avascular tumors are complex and poorly understood. In this paper, we investigate the formation of a necrotic core in a 3D tumor cell culture within a microfluidic device, considering oxygen, nutrients, and the microenvironment acidification by means of a computational-mathematical model. Our objective is to simulate cell processes, including proliferation and death inside a microfluidic device, according to the microenvironmental conditions. We employed approximation utilizing finite element models taking into account glucose, oxygen, and hydrogen ions diffusion, consumption and production, as well as cell proliferation, migration and death, addressing how tumor cells evolve under different conditions. The resulting mathematical model was examined under different scenarios, being capable of reproducing cell death and proliferation under different cell concentrations, and the formation of a necrotic core, in good agreement with experimental data reported in the literature. This approach not only advances our fundamental understanding of necrotic core formation but also provides a robust computational platform to study personalized therapeutic strategies, offering an important tool in cancer research and treatment design.

Bacteria/Nanozyme Composites: New Therapeutics for Disease Treatment.

Bacterial therapy is recognized as a cost-effective treatment for several diseases. However, its development is hindered by limited functionality, weak inherent therapeutic effects, and vulnerability to harsh microenvironmental conditions, leading to suboptimal treatment activity. Enhancing bacterial activity and therapeutic outcomes emerges as a pivotal challenge. Nanozymes have garnered significant attention due to their enzyme-mimic activities and high stability. They enable bacteria to mimic the functions of gene-edited bacteria expressing the same functional enzymes, thereby improving bacterial activity and therapeutic efficacy. This review delineates the therapeutic mechanisms of bacteria and nanozymes, followed by a summary of strategies for preparing bacteria/nanozyme composites. Additionally, the synergistic effects of such composites in biomedical applications such as gastrointestinal diseases and tumors are highlighted. Finally, the challenges of bacteria/nanozyme composites are discussed and propose potential solutions. This study aims to provide valuable insights to offer theoretical guidance for the advancement of nanomaterial-assisted bacterial therapy.

A pH-responsive single-atom nanozyme for photothermal-augmented nanocatalytic tumor therapy

Based on the physiological conditions of the tumor microenvironment (TME), many effective therapeutic strategies have been reported by using nanocatalysts. The main challenge is the insufficient catalytic activity of nanocatalysts within the acidic TME, significantly constraining their therapeutic efficacy. Herein, a pH-responsive bifunctional platform is developed with multiple enzyme-like catalytic activities for synergistic tumor therapy by integrating Rh single atoms nanozymes (SA-Rh nanozymes) with photothermal therapy (PTT). The SA-Rh nanozymes display peroxidase-mimicking activities within tumor cells, inducing a synergistic effect of enhanced reactive oxygen species generation for collaborative cancer therapy involving both chemodynamic therapy and PTT. Additionally, SA-Rh nanozymes exhibit catalase mimicking activities, enabling the generation of oxygen, and facilitating efficient nanozyme catalytic therapy in a substrate-cycle manner. Moreover, the catalytic efficiency of SA-Rh nanozymes is found to be higher under weak acidic conditions (pH=6.0) compared to neutral conditions (pH=7.4), thereby enabling the maintenance of catalytic activity in an acidic environment. The incorporation of this feature enhances its catalytic activity within the TME, optimizing the efficacy of nanozyme. The remarkable near-infrared I region absorption capability confers SA-Rh nanozymes with exceptional PTT performance, achieving an impressive efficiency of 34.1 %. Therefore, the SA-Rh nanozymes exhibit a remarkable ability to induce apoptosis in cancer cells through synergistic CDT and PTT modalities, effectively overcoming the shortcomings of current nanozyme catalytic therapy.

Enhancing Magnetic Hyperthermia Efficacy through Targeted Heat Shock Protein 90 Inhibition: Unveiling Immune-Mediated Therapeutic Synergy in Glioma Treatment.

The induction of heat stress response (HSR) mediated by the generation of heat shock proteins (HSPs) on exposure to magnetic hyperthermia-mediated cancer therapy (MHCT) decreases the efficacy of localized heat treatment at the tumor site, and thus therapy remains a significant challenge. Hence, the present study examined differential HSR elicited in glioma cells post-MHCT under different tumor microenvironment conditions (2D monolayers, 3D monoculture, and coculture spheroids) to recognize target genes that, when downregulated, could enhance the therapeutic effect of MHCT. Gene expression analysis following MHCT revealed that HSP90 was upregulated as compared to HSP70. Hence, to enhance the efficacy of the treatment, a combinatorial strategy using 17-DMAG as an inhibitor of HSP90 following MHCT was investigated. The effects of combinatorial therapy in terms of cell viability, HSP levels by immunofluorescence and gene expression analysis, oxidative stress generation, and alterations in cellular integrity were evaluated, where combinatorial therapy demonstrated an enhanced therapeutic outcome with maximum glioma cell death. Further, in the murine glioma model, a rapid tumor inhibition of 65 and 53% was observed within 8 days at the primary and secondary tumor sites, respectively, in the MCHT + 17-DMAG group, with abscopal effect-mediated complete tumor inhibition at both the tumor sites within 20 days of MHCT. The extracellularly released HSP90 from dying tumor cells further suggested the induction of immune response supported by the upregulation of IFN-γ and calreticulin genes in the MHCT + 17-DMAG group. Overall, our findings indicate that MHCT activates host immune systems and efficiently cooperates with the HSP90 blockade to inhibit the growth of distant metastatic tumors.

MDB-35. DEVELOPMENT OF DUAL LUCIFERASE ASSAY FOR 3D-COMPATIBLE HIGH THROUGHPUT SCREENING IN MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the most common malignant pediatric brain tumor, originating from the cerebellum, a part of the brain that controls balance, coordinated movement and other non-motor behaviors. 2D cell cultures are commonly used for testing of new compounds in high throughput screening (HTS) targeting this cancer, largely because there are numerous highly optimized assays available for analyzing this cell culture model. However, they have significant limitation in mimicking in vivo conditions of the tumor microenvironment. 3D cell-culture methods have been embraced to enhance the modeling of in vivo tissue, replicating the multicellular architecture and the characteristics of the extracellular matrix. Presently, there are efforts to integrate 3D cell-culture with HTS to yield more clinically relevant results. However, one of the requirements for this is a 3D-compatible cell-based assay suitable for HTS. We developed such an assay which measures the activity of YAP/TAZ, a central molecule of Hippo-signaling pathway, and has been implicated in medulloblastoma. It plays a crucial role in maintaining tissue balance by coordinating proliferation, apoptosis, and differentiation. METHODS We cloned 8xGTIIC-NlucP, a brighter and more sensitive luciferase (Nanoluciferase) reporter plasmid that detects YAP/TAZ activity. Responsiveness to YAP/TAZ activity, and its inhibition were detected using a dual luciferase assay in the medulloblastoma cell lines after transfection. A normalization plasmid with a constitutive promoter for firefly luciferase was used in this assay to differentiate compounds that affect YAP/TAZ activity specifically as opposed to those that are toxic to the cells. RESULTS A robust signal for YAP/TAZ activity, and its reduction when cells were treated with inhibitor, was seen in the three medulloblastoma cell lines used, which further validates the result. CONCLUSION In conclusion, we were able to develop a 3D-compatible cell-based assay with Nanoluciferase which can be a foundation for HTS targeting medulloblastoma using 3D cultures.

Disentangling the roles of bracken fronds and litter on natural seedling recruitment in fire-disturbed tropical montane habitats

Tropical montane forests are diverse ecosystems often affected by uncontrolled human-induced fires causing tree mortality and creating large deforested areas. After fires, Pteridium spp. ferns (bracken) often dominate, and forest regeneration in these areas is slow. In this study, we evaluated the effects of bracken fronds and litter, as well as the micro-environmental conditions created by the fern, on the density and species diversity of naturally recruiting seedlings. At eight sites, 120 experimental plots were established among forest and bracken-dominated areas with the following treatments: (a) fronds and litter intact (F+L+); (b) fronds intact and litter removed (F+L-); (c) fronds removed and litter intact (F-L+); and (d) fronds and litter removed (F-L-). After one year, all seedlings were registered, identified and classified according to their life-form (tree, shrub, herb, vine), dispersal vector (wind- or animal-dispersed) and successional status (early-, mid-, and late-successional). For all treatments we assessed 12 micro-environmental variables. We identified 3649 naturally-recruiting seedlings corresponding to 278 species from 70 families. We found positive effects of bracken fronds particularly on tree seedling recruitment: treatments with fronds had greater densities of both animal- and wind-dispersed tree seedlings, 1.8 and 1.4 fold higher, respectively, compared to treatments without fronds. Similarly, the density of early-, mid- and late-successional tree species was 1.3, 1.7 and 1.9 times higher in treatments with than without fronds. Furthermore, species diversity of early-, mid- and late-successional tree species was higher in the treatments with fronds. The environmental conditions generated by bracken presence, such as photosynthetically active radiation, soil temperature, live bracken biomass and litter depth, had positive effects on seedling density of all, animal-dispersed and early-successional tree species, and negative effects on seedling recruitment of other life-forms, notably from early successional stages. To promote forest regeneration in bracken-dominated areas, active restoration measures such as direct seed addition and transplants of nursery-raised seedlings of mid- and late-successional species should be considered. Since bracken can hinder the establishment of some life forms and species but favor others, it is recommended to include a trait-based approach to understand species responses to environmental factors and select species that are optimally adapted to bracken-dominated habitat conditions to promote forest regeneration.

Agent-based model demonstrates the impact of nonlinear, complex interactions between cytokinces on muscle regeneration.

Muscle regeneration is a complex process due to dynamic and multiscale biochemical and cellular interactions, making it difficult to identify microenvironmental conditions that are beneficial to muscle recovery from injury using experimental approaches alone. To understand the degree to which individual cellular behaviors impact endogenous mechanisms of muscle recovery, we developed an agent-based model (ABM) using the Cellular-Potts framework to simulate the dynamic microenvironment of a cross-section of murine skeletal muscle tissue. We referenced more than 100 published studies to define over 100 parameters and rules that dictate the behavior of muscle fibers, satellite stem cells (SSCs), fibroblasts, neutrophils, macrophages, microvessels, and lymphatic vessels, as well as their interactions with each other and the microenvironment. We utilized parameter density estimation to calibrate the model to temporal biological datasets describing cross-sectional area (CSA) recovery, SSC, and fibroblast cell counts at multiple timepoints following injury. The calibrated model was validated by comparison of other model outputs (macrophage, neutrophil, and capillaries counts) to experimental observations. Predictions for eight model perturbations that varied cell or cytokine input conditions were compared to published experimental studies to validate model predictive capabilities. We used Latin hypercube sampling and partial rank correlation coefficient to identify in silico perturbations of cytokine diffusion coefficients and decay rates to enhance CSA recovery. This analysis suggests that combined alterations of specific cytokine decay and diffusion parameters result in greater fibroblast and SSC proliferation compared to individual perturbations with a 13% increase in CSA recovery compared to unaltered regeneration at 28 days. These results enable guided development of therapeutic strategies that similarly alter muscle physiology (i.e. converting extracellular matrix [ECM]-bound cytokines into freely diffusible forms as studied in cancer therapeutics or delivery of exogenous cytokines) during regeneration to enhance muscle recovery after injury.