Genomic and single-cell analyses reveal genetic signatures of swimming pattern and diapause strategy in jellyfish.

Jellyfish exhibit innovative swimming patterns that contribute to exploring the origins of animal locomotion. However, the genetic and cellular basis of these patterns remains unclear. Herein, we generated chromosome-level genome assemblies of two jellyfish species, Turritopsis rubra and Aurelia coerulea, which exhibit straight and free-swimming patterns, respectively. We observe positive selection of numerous genes involved in statolith formation, hair cell ciliogenesis, ciliary motility, and motor neuron function. The lineage-specific absence of otolith morphogenesis- and ciliary movement-related genes in T. rubra may be associated with homeostatic structural statocyst loss and straight swimming pattern. Notably, single-cell transcriptomic analyses covering key developmental stages reveal the enrichment of diapause-related genes in the cyst during reverse development, suggesting that the sustained diapause state favours the development of new polyps under favourable conditions. This study highlights the complex relationship between genetics, locomotion patterns and survival strategies in jellyfish, thereby providing valuable insights into the evolutionary lineages of movement and adaptation in the animal kingdom.

Pollution level and ecological risk assessment of triazine herbicides in Laizhou Bay and derivation of seawater quality criteria.

Triazine herbicides are widely used in agriculture and have become common pollutants in marine environments. However, the spatiotemporal distribution characteristics and water quality criteria (WQC) of triazine herbicides are still unclear. This study found that triazine herbicides had a high detection rate of 100 % in surface seawater of Laizhou Bay, China, with average concentrations of 217.61, 225.13, 21.97, and 1296.72 ng/L in March, May, August, and October, respectively. Moreover, estuaries were important sources, and especially the Yellow River estuary exhibited the highest concentrations of 16,115.86 ng/L in October. The 10 triazine herbicides were detected in the sediments of Laizhou Bay, with a concentration ranging from 0.14-1.68 µg/kg. Atrazine and prometryn accounted for 33.41 %-59.10 % and 28.93 %-50.06 % of the total triazine herbicides in the seawater, and prometryn had the highest proportion (63.50 %) in the sediments. Correlation analysis revealed that triazine herbicides led to the loss of plankton biodiversity, which further decreased the dissolved oxygen. In addition, this study collected 45 acute toxicity data and 22 chronic toxicity data of atrazine, 16 acute toxicity data of prometryn, and supplemented with toxicity experiments of prometryn on marine organisms. Based on the toxicity database, the WQCs of atrazine and prometryn were derived using species sensitivity distribution. The overall risk probability of atrazine and prometryn were both less than 1.75 % in the Laizhou Bay, indicating an acceptable risk. This study not only clarified the pollution status and ecological risk of triazine herbicides, but also provided scientific basis for their environmental management standards.

Implications of ocean warming and acidification on heavy metals in surface seawater of the Bohai Sea.

At present, a clear dependency of the dynamics upon temperature and pH has not been established for many heavy metals (HMs), so making it difficult to project and quantify the impact of ocean warming and acidification on metal biogeochemistry in future scenarios. To understand the responses of HMs to future ocean warming and acidification, we estimated the spatial-temporal variations and pollution status of six dissolved HMs (i.e., Cu, Zn, Pb, Cd, Hg, and As) in surface seawater throughout the Bohai Sea during 2012-2014. The results showed that the average concentrations of Cu, Zn, Pb, Cd, Hg, and As in seawater of the Bohai Sea were between 2.01-3.18, 10.47-15.58, 0.85-2.31, 0.25-0.55, 0.05-0.13, and 1.24-1.98 µg L-1, respectively. Spatially, the average concentrations of the studied HMs generally decreased from the three bays towards the central area, except for Hg which was relatively high in the central Bohai Sea in some cases. This implied that, in addition to continental inputs, there may be other processes affecting the distribution pattern of Hg, such as cyclonic or anticyclonic gyres, benthic fluxes between surface and bottom layers, and some marine planktonic and microbial activities. The pollution assessments of six HMs in seawater revealed that the major risk pollutants were Pb and Hg across the Bohai Sea. Analyses of the local and interactive effects of temperature and pH on HMs showed that the interactive effect of changing temperature and pH on HMs is much more complex than a direct temperature/pH relationship with HMs. Altogether, the results suggested that future ocean warming and acidification will significantly influence the concentrations of dissolved HMs in seawater of the Bohai Sea, but with different relationships.

Unconventional mechanism of action and resistance to rapalogs in renal cancer.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

Impact of summer hypoxia on macrobenthic communities in a semi-enclosed bay: A long-term observation in the North Yellow sea of China.

The O2 content of the global ocean has been declining progressively over the past decades, mainly because of human activities and global warming. Despite this situation, the responses of macrobenthos under hypoxic conditions remain poorly understood. In this study, we conducted a long-term observation (2015-2022) to investigate the intricate impact of summer hypoxia on macrobenthic communities in a semi-enclosed bay of the North Yellow Sea. Comparative analyses revealed higher macrobenthos abundance (1956.8 ± 1507.5 ind./m2 vs. 871.8 ± 636.9 ind./m2) and biomass (8.2 ± 4.1 g/m2 vs. 5.6 ± 3.2 g/m2) at hypoxic sites compared to normoxic sites during hypoxic years. Notably, polychaete species demonstrated remarkable adaptability, dominating hypoxic sites, and shaping community structure. The decline in biodiversity underscored the vulnerability and diminished resilience of macrobenthic communities to hypoxic stressors. Stable isotope analysis provided valuable insights into food web structures. The average trophic level of macrobenthos measured 2.84 ± 0.70 at hypoxic sites, contrasting with the higher value of 3.14 ± 0.74 observed at normoxic sites, indicating the absence of predators at high trophic levels under hypoxic conditions. Moreover, trophic interactions were significantly altered, resulting in a simplified and more vulnerable macrobenthic trophic structure. The findings underscored the importance of comprehensive research to understand the complex responses of macrobenthic communities to hypoxia, thereby informing future conservation efforts in impacted ecosystems.

Typhoon-induced stormwater drives nutrient dynamics and triggers phytoplankton blooms in Laizhou Bay, China.

In this study, we investigated the hydrological and ecological impacts of heavy rainfall caused by the storm Rumbia and Typhoon Lekima on Laizhou Bay (LZB) through land‒sea synchronous field surveys, online remote sensors, and simulated enclosure experiments. Within two weeks of Rumbia, approximately 9% and 16% of the annual riverine total nitrogen (TN) and total phosphorus (TP) fluxes, respectively, were transported to the LZB and the proportions were 17% and 35%, respectively, for Lekima. The land use on the watersheds increased the rates of land-derived nutrient loading and altered their biogeochemical forms. Consequently, the average concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) in the LZB increased by 2.6 and 1.0 times post-Rumbia and by 3.5 and 1.3 times post-Lekima, respectively. Relatively lower salinity and temperature, sudden increases in DIN, and strengthened coastal currents stimulated the growth of highly adaptable and small diatoms, resulting in the first diatom blooms. Subsequently, a bloom of Noctiluca scintillans formed.

Biodiversity and distribution patterns of blooming jellyfish in the Bohai Sea revealed by eDNA metabarcoding.

BACKGROUND: The mass occurrence of scyphozoan jellyfish severely affects marine ecosystems and coastal economies, and the study of blooming jellyfish population dynamics has emerged in response. However, traditional ecological survey methods required for such research have difficulties in detecting cryptic life stages and surveying population dynamics owing to high spatiotemporal variations in their occurrence. The environmental DNA (eDNA) technique is an effective tool for overcoming these limitations. RESULTS: In this study, we investigated the biodiversity and spatial distribution characteristics of blooming jellyfish in the Bohai Sea of China using an eDNA metabarcoding approach, which covered the surface, middle, and bottom seawater layers, and sediments. Six jellyfish taxa were identified, of which Aurelia coerulea, Nemopilema nomurai, and Cyanea nozakii were the most dominant. These three blooming jellyfish presented a marked vertical distribution pattern in the offshore regions. A. coerulea was mainly distributed in the surface layer, whereas C. nozakii and N. nomurai showed a upper-middle and middle-bottom aggregation, respectively. Horizontally, A. coerulea and C. nozakii were more abundant in the inshore regions, whereas N. nomurai was mainly distributed offshore. Spearman's correlation analysis revealed a strong correlation between the eDNA of the three dominant blooming jellyfish species and temperature, salinity, and nutrients. CONCLUSIONS: Our study confirms the applicability of the eDNA approach to both biodiverstiy evaluation of blooming jellyfish and investigating their spatial distribution, and it can be used as a supplementary tool to traditional survey methods.

Application of environmental DNA metabarcoding and quantitative PCR to detect blooming jellyfish in a temperate bay of northern China.

Frequently occurring jellyfish blooms have severe impacts on the socioeconomics of coastal areas, which stress the importance of early detection and assessments of blooming jellyfish taxa. Environmental DNA (eDNA) techniques (quantitative PCR and eDNA metabarcoding) have the advantage of high sensitivity and are an emerging powerful tool for investigations of target species. However, a comprehensive analysis of the biodiversity and biomass of jellyfish taxa in the target area by combining the two eDNA techniques is still lacking. Here, we developed eDNA metabarcoding and quantitative PCR for the detection and assessment of jellyfish taxa in the temperate Yantai Sishili Bay (YSB) and estimated the spatial distribution of Aurelia coerulea. Species-specific quantitative PCR assays targeting the mitochondrial cytochrome c oxidase subunit I gene of A. coerulea were developed. Additionally, eDNA metabarcoding based on the mitochondrial 16S rDNA sequences identified six jellyfish species in YSB. Moreover, our results indicate that A. coerulea aggregations were more likely to occur in the inner part of the bay than in the outer part, and they gathered in the bottom layer of seawater rather than in the surface layer. Our results demonstrate the potential of two eDNA techniques in jellyfish biomass investigation and jellyfish taxa detection. These eDNA techniques may contribute to the discovery of jellyfish aggregation so as to achieve early warning of large-scale jellyfish blooms in coastal areas.

Efficiency and ecological safety of herbicide haloxyfop-R-methyl on removal of coastal invasive plant Spartina alterniflora.

Spartina alterniflora is a global invasive plant and has caused considerable damage to coastal wetland ecosystem. This study evaluated the efficiency and ecological safety of herbicide haloxyfop-R-methyl (HPME) in removing S alterniflora in Laizhou Bay. The results showed that the density of regenerated S. alterniflora after 10 months of application of 0.01, 0.02 and 0.03 g/m2 HPME decreased by 86.67 %, 99.16 % and 99.31 %, respectively. Moreover, seed abortion rates were 62.25 %, 92.24 % and 94.82 %, and weight of roots in HPME groups were 56.63 %, 59.99 %, and 40.10 % of those in the control group. After 4 days of application, HPME could not be detected in S. alterniflora and sediments. In addition, HPME did not change sediment physicochemical properties, macrozoobenthos community and microbial community structure during 16 days, but increased the density of native macrozoobenthos after 1 year. Therefore, HPME might be an effective and ecologically safe chemical for the eradication of S. alterniflora.

Distribution and pollution assessment of marine debris off-shore Shandong from 2014 to 2022.

This study analysed marine debris monitoring data for Shandong from 2014 to 2022 to obtain a better understanding of marine debris stocking off-shore Shandong in order to reduce marine debris pollution and improving the ecological environment of ocean. The results indicated that the abundance of coastal marine debris was 45,832 items/km2 (1118.5 kg/km2); the abundance of small/medium sized floating marine debris was 8976 items/km2 (1.38 kg/km2); and the abundance of large floating marine debris was 35 items/km2; the abundance of seafloor debris was 104 item/km2 (0.22 kg/km2). Compared with the nationwide abundance of marine debris, the quantity density of floating marine debris in Shandong was higher; the abundance of coastal marine debris and quality density of floating marine debris were lower. The majority of the Shandong marine debris was small/medium plastic, mostly from human activities. And we found no significant correlation between precipitation and the abundance of marine debris by statistical analysis.

Electrochemical fluorescence switching of enhanced green fluorescent protein.

Switchable fluorescent proteins, for which fluorescence can be switched ON and OFF, are widely used for molecule tracking and super resolution imaging. However, the robust use of the switchable fluorescent proteins is still limited as either the switching is not repeatable, or such switching requires irradiation with coupled lasers of different wavelengths. Herein, we report an electrochemical approach to reversible fluorescence switching for enhanced green fluorescent proteins (EGFP) on indium tin oxide coated glass. Our results demonstrate that negative and positive electrochemical potentials can efficiently switch the fluorescent proteins between the dim (OFF) and bright (ON) states at the single molecule level. The electrochemical fluorescence switching is fast, reversible, and may be performed up to hundreds of cycles before photobleaching occurs. These findings highlight that this method of electrochemical fluorescence switching can be incorporated into advanced fluorescence microscopy.

Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse.

Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.

Carbon sequestration from refractory dissolved organic carbon produced by biodegradation of Saccharina japonica.

Using macroalgae cultures to sequester carbon has been proposed in recent years. Yet the key mechanism of carbon sequestration-how carbon in degrading biomass is converted into refractory dissolved organic carbon (RDOC) remains poorly understood. The process of producting RDOC via biomass degradation of Saccharina japonica, the most productive algae in China, was thus studied in the laboratory. Most of the carbon in the kelp biomass was converted to dissolved inorganic carbon (DIC) by bacterial respiration. Only 7.8% of the carbon in the kelp biomass was converted into labile DOC, semi-labile or semi-refractory DOC, and refractory DOC in turn. The enrichment of DIC resulted in hypoxic conditions in the water. For the hypoxia in the experiment, the sulfur-degrading bacteria Campylobacteria and Bacteroidia became the dominant bacterial classes, which were the key drivers for the transformation of labile DOC to semi-labile or semi-refractory DOC. Then, semi-labile or semi-refractory DOC was converted to RDOC, driven by the sulfite-reducing bacteria Clostridia and Kapabacteria. Finally, 0.3% of the carbon content in kelp was transformed into RDOC. The resulting RDOC, which was rich in sulfur and nitrogen elements, increased the molecular diversity and average molecular weight in the water. An important finding was that the production of RDOC may be accompanied by the environmental risk of hypoxia.

HIF2 Inactivation and Tumor Suppression with a Tumor-Directed RNA-Silencing Drug in Mice and Humans.

PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that ∼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.

[Spatial Distribution and Eco-stoichiometric Characteristics of Soil Nutrient Elements Under Different Vegetation Types in the Yellow River Delta Wetland].

To clarify the distribution characteristics and the ecological stoichiometric characteristics of nutrient elements in soils under different vegetation types, four typical natural wetlands, i.e., Phragmites australis wetland, Tamarix chinensis wetland, Suaeda salsa wetland, and Tidal flat wetland, as well as Gossypium spp. fields that were reclaimed from natural wetlands, were selected as study sites in the Yellow River Delta, and comparisons between the agricultural reclamation land and natural wetlands were conducted. The results showed that the soil total organic carbon (TOC) and total nitrogen (TN) contents in the natural wetlands were as follows:P. australis wetland and T. chinensis wetland>S. salsa wetland>Tidal flat, and the contents of TOC and TN were significantly negatively related to electrical conductivity (EC) and pH values (P<0.05). The contents of TOC, TN, and total phosphorus (TP) in Gossypium spp. fields were significantly higher than those in natural wetlands (P<0.05), especially the contents of nitrate nitrogen (NO3--N) in Gossypium spp. fields, which were 9.4-11.4 times that of natural wetlands. However, no significant correlations between TOC, TN, and TP and EC and pH values (P>0.05) were observed in Gossypium spp. fields. The results of correlation analysis showed that the C/N of natural wetlands were mainly controlled by the contents of TN (P<0.05), and the C/N of the Gossypium spp. fields were significantly lower than those of natural wetlands (P<0.05). The soil C/P and N/P of natural wetlands and Gossypium spp. fields in the Yellow River Delta were low, and the variation trends were consistent with those of soil TOC and TN. Comparative analysis revealed, on the whole, that there were significantly different soil nutrient element contents, C/N, C/P, and N/P in Gossypium spp. fields compared to those of natural wetlands (P<0.05). The process of reclamation could significantly change the spatial distribution of nutrient elements in wetlands. Our results should be of importance in revealing the biogeochemical process of soil nutrient elements in coastal wetland and the influence of agricultural reclamation activities on the differentiation of soil nutrient elements.

Lifetime based axial contrast enable simple 3D-STED imaging.

Stimulated Emission Depletion (STED) microscopy increase spatial image resolution by laterally sharpening the illumination profile of the confocal microscope. However, it remains compromised in axial resolution. To improve axial STED resolution, constructive interference of the STED depletion beam must be formed surrounding the focal plane to turn off the fluorophores beyond the focal plane. For isotropic 3D-STED resolution, this axial STED interference pattern must be overlayed with the doughnut STED beam at nanometer accuracy. Such optical configurations can be challenging in alignment. In this current work, we introduced a straightforward lifetime based axial contrast in STED microscope by imaging the samples on an ITO coated glass coverslip. The STED laser generates surface plasmon resonance on the ITO surface that enhanced the metal induced energy transfer MIET effect on the ITO surface. The enhanced MIET effect established a lifetime gradient with ∼20% dynamic range that extend for mor than 400 nm from the ITO surface. The axial contrast based on the lifetime gradient was directly used for 3D-STED imaging of tubulin fibers inside COS-7 cells, where the vertical displacement of single tubulin fiber was revealed. Lifetime gating could be applied to further improve lateral spatial resolution. Considering that most common implementation of STED microscopes uses pulsed lasers and timing electronics, there is no optical modification of the microscope is required in the current 3D-STED approach.

Integration of chemical and biological methods: A case study of polycyclic aromatic hydrocarbons pollution monitoring in Shandong Peninsula, China.

Polycyclic aromatic hydrocarbons (PAHs), as persistent toxic substances (PTS), have been widely monitored in coastal environment, including seawater and sediment. However, scientific monitoring methods, like ecological risk assessment and integrated biomarker response, still need massive researches to verify their availabilities. This study was performed in March, May, August and October of 2018 at eight sites, Yellow River estuary (S1), Guangli Port (S2), Xiaying (S3), Laizhou (S4), Inner Bay (S5), Outer Bay (S6), Hongdao (S7) and Hongshiya (S8) of Shandong Peninsula, China. The contents of 16 priority PAHs in local seawater and sediment were determined, by which ecological risk assessment risk quotient (RQ) for seawater and sediment quality guidelines (SQGs) were calculated to characterize the PAHs pollution. Meanwhile, multiple biomarkers in the digestive gland of clam Ruditapes philippinarum were measured to represent different biological endpoints, including ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), sulfotransferase (SULT), superoxide dismutase (SOD) and lipid peroxidation (LPO), by which integrated biomarker response (IBR) was calculated to provide a comprehensive assessment of environmental quality. Taken together, these results revealed the heaviest pollution at S2 as both PAHs concentrations and biomarkers responses reflected, and supported the integrated biomarker response as a useful tool for marine environmental monitoring, through its integration with SQGs.

An integrated approach using chemical ecological risk assessment and multi-integrated biomarker indexes approach to assess pollution: A case study of Ruditapes philippinarum in four bays on the Shandong Peninsula in China.

Considering the ecological risks of polycyclic aromatic hydrocarbons (PAHs) to the marine environment, it is urgent to find scientific and effective monitoring methods. In this study, an integrated approach combining chemical ecological risk assessment and multi-integrated biomarker indexes approach was used to assess the marine environment. Samples included seawater, sediments, and clam Ruditapes philippinarum were collected from four bays on the Shandong Peninsula, China in the four seasons of 2019. The concentrations, composition, potential sources, and ecological risk of PAHs were investigated in seawater and sediments. Risk quotient (RQ) and sediment quality guidelines (SQGs) were calculated to assess the ecological risks of PAHs in seawater and sediment, respectively. And then, clam Ruditapes philippinarum's multi-level biological response, including its ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), superoxide dismutase (SOD), lipid peroxidation (LPO), and acetylcholinesterase (AChE) were investigated in-depth, by which multi-integrated biomarker indexes approach were calculated to evaluate marine environmental quality. Taken together, the results showed that the concentration of PAHs was in good agreement with the response of biomarkers, and the usefulness of the combined use of chemical ecological risk assessment and integrated biomarker indexes to assess PAHs pollution was verified.

Determinants of renal cell carcinoma invasion and metastatic competence.

Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.