Compression drives diverse transcriptomic and phenotypic adaptations in melanoma.

Physical forces are prominent during tumor progression. However, it is still unclear how they impact and drive the diverse phenotypes found in cancer. Here, we apply an integrative approach to investigate the impact of compression on melanoma cells. We apply bioinformatics to screen for the most significant compression-induced transcriptomic changes and investigate phenotypic responses. We show that compression-induced transcriptomic changes are associated with both improvement and worsening of patient prognoses. Phenotypically, volumetric compression inhibits cell proliferation and cell migration. It also induces organelle stress and intracellular oxidative stress and increases pigmentation in malignant melanoma cells and normal human melanocytes. Finally, cells that have undergone compression become more resistant to cisplatin treatment. Our findings indicate that volumetric compression is a double-edged sword for melanoma progression and drives tumor evolution.

Cytoskeletal tension actively sustains the migratory T-cell synaptic contact.

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

Biophysical informatics reveals distinctive phenotypic signatures and functional diversity of single-cell lineages.

MOTIVATION: In this work, we present an analytical method for quantifying both single-cell morphologies and cell network topologies of tumor cell populations and use it to predict 3D cell behavior. RESULTS: We utilized a supervised deep learning approach to perform instance segmentation on label-free live cell images across a wide range of cell densities. We measured cell shape properties and characterized network topologies for 136 single-cell clones derived from the YUMM1.7 and YUMMER1.7 mouse melanoma cell lines. Using an unsupervised clustering algorithm, we identified six distinct morphological subclasses. We further observed differences in tumor growth and invasion dynamics across subclasses in an in vitro 3D spheroid model. Compared to existing methods for quantifying 2D or 3D phenotype, our analytical method requires less time, needs no specialized equipment and is capable of much higher throughput, making it ideal for applications such as high-throughput drug screening and clinical diagnosis. AVAILABILITY AND IMPLEMENTATION: https://github.com/trevor-chan/Melanoma_NetworkMorphology. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Translational medicine for acute lung injury.

Acute lung injury (ALI) is a complex disease with numerous causes. This review begins with a discussion of disease development from direct or indirect pulmonary insults, as well as varied pathogenesis. The heterogeneous nature of ALI is then elaborated upon, including its epidemiology, clinical manifestations, potential biomarkers, and genetic contributions. Although no medication is currently approved for this devastating illness, supportive care and pharmacological intervention for ALI treatment are summarized, followed by an assessment of the pathophysiological gap between human ALI and animal models. Lastly, current research progress on advanced nanomedicines for ALI therapeutics in preclinical and clinical settings is reviewed, demonstrating new opportunities towards developing an effective treatment for ALI.

GWAS of biological aging to find longevity genes in schizophrenia.

Schizophrenia (SCZ) is a severe psychotic disorder associated with premature mortality and aging. Moreover, the symptoms and progression of psychiatric disorders in general are associated with decreased lifespan, biological aging, and poorer medical outcomes. In this study, we investigated the relationship between several epigenetic clocks and scanned the entire genome for association in a cohort of SCZ individuals (n = 107). Biological age was computed from blood DNA methylation (DNAm) and tested for association against  common  variants across the genome using general linear models. Genes affecting epigenetic age acceleration in our cohort were found mainly when using the telomeric length clock rather than the other biological clocks. These findings pair with existing evidence that there are some genes associated with longevity and suggest further investigations of  putative biological mechanisms for morbidity and premature mortality, not only in patients with SCZ but also in the general population.

Visualization of microRNA therapy in cancers delivered by small extracellular vesicles.

MicroRNA (miRNA) delivery by extracellular vesicles (EVs) has recently inspired tremendous developments in cancer treatments. However, hybridization between miRNA and its target mRNA is still difficult to be imaged in vivo to assess the therapeutic effects in time. Herein we design a nano-scale fluorescent "off-on" complex encapsulated by small extracellular vesicles (sEVs) for real-time visualization and evaluation of gene therapy efficiency in human gastric cancer cells and murine xenograft tumor models. The complex is formed by π-π stacking between graphene quantum dots (GQDs) and tumor suppressor miR-193a-3p conjugated fluorescent tag whose signals remain off when binding to GQDs. Loaded into sEVs using tunable sonication techniques, the GQDs/Cy5-miR particles enter the tumor cells and promote miR-193a-3p escape from endosomes. The miR-193a-3p in GQDs/Cy5-miR is unleashed to pair the specific target oncogene cyclin D1 (CCND1), therefore turning on the fluorescence of miRNA tags. We find out that GQDs/Cy5-miR@sEVs can activate the "turn-on" fluorescent signal and exhibit the longest retention time in vivo, which suggests a minimized degradation of miR-193a-3p in dynamic processes of miRNA-mRNA binding. More importantly, GQDs/Cy5-miR@sEVs significantly promote cancer apoptosis in vitro and in vivo via the enhanced cellular uptake. Our study demonstrates that GQDs/Cy5-miR@sEVs represent an efficient and refined theranostic platform for gene therapy in cancers.

Biophysical Informatics Approach For Quantifying Phenotypic Heterogeneity In Cancer Cell Migration In Confined Microenvironments.

MOTIVATION: Cancer cell heterogeneity can manifest genetically and phenotypically. Bioinformatics methods have been used to analyze complex genomics and transcriptomics data, but have not been well-established for analyzing biophysical data of phenotypically heterogeneous tumor cells. Here, we take an informatics approach to analyze the biophysical data of MDA-MB-231 cells, a widely used breast cancer cell line, during their spontaneous migration through confined environments. Experimentally, we vary the constriction microchannel geometries (wide channel, short constriction, and long constriction) and apply drug treatments. We find that cells in the short constriction are similar in morphology to the cells in the wide channel. However, their fluorescence profiles are comparable to those in the long constriction. We demonstrate that the cell migratory phenotype is correlated more to mitochondria in a non-confined environment and more to actin in a confined environment. We demonstrate that the cells' migratory phenotypes are altered by ciliobrevin D, a dynein inhibitor, in both confined and non-confined environments. Overall, our approach elucidates phenotypic heterogeneity in cancer cells under confined microenvironments at single-cell resolution. RESULTS: Here, we apply a bioinformatics approach to a single cell invasion assay. We demonstrate that this method can determine distinctions in morphology, cytoskeletal activities, and mitochondrial activities under various geometric constraints and for cells of different speeds. Our approach can be readily adapted to various heterogeneity studies for different types of input biophysical data. In addition, this approach can be applied to studies related to biophysical changes due to differences in external stimuli, such as treatment effects on cellular and subcellular activities, at single-cell resolution. Finally, as similar bioinformatics methods have been widely applied in studies of genetic heterogeneity, biophysical information extracted using this approach can be analyzed together with the genetic data to relate genetic and phenotypic heterogeneity. AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MRI of the Carpal Tunnel 3 and 12 Months After Endoscopic Carpal Tunnel Release.

OBJECTIVE. The purpose of this study was to study changes in the median nerve, retinaculum, and carpal tunnel on MRI after successful endoscopic carpal tunnel release (ECTR). SUBJECTS AND METHODS. In this prospective study, 35 wrists in 32 patients (five men, 27 women; mean age, 56.7 ± 6.8 [SD] years) with nerve conduction test-confirmed primary carpal tunnel syndrome were evaluated from May 2013 to September 2016. Clinical scores ranging from 0 to 4 (no improvement to symptoms completely resolved) and MRI morphologic features of median nerve and carpal tunnel were evaluated at baseline and 3 and 12 months after ECTR. The paired t test was used to compare MRI parameters before and after ECTR and their relationships to clinical improvement scores. RESULTS. All patients' conditions improved after ECTR with mean clinical improvement scores of 2.94 ± 1.0 at 3 months and 3.49 ± 0.56 at 12 months. Although median nerve swelling did decrease proximally, the nerve remained swollen (> 15 mm2) and flattened in all areas, even 12 months after ECTR. Additional changes occurred in median nerve caliber-change ratio, relative signal intensity, and carpal tunnel cross-sectional area. A retinacular gap was present in 33 (94%) wrists 3 months and six (17%) wrists 12 months after ECTR, and increased retinacular bowing persisted. CONCLUSION. After ECTR, undue swelling and flattening of the median nerve persist as long as 12 months after surgery, even in patients with a good surgical outcome. One should be wary of using these MRI findings as signs of persistent neural compression. The retinaculum reforms in most patients within 12 months of surgery but with a more bowed configuration.

Neurovascular Island Flap for Pulp and Nail Augmentation in Thumb Duplication Reconstruction: A Surgical Method With Long-Term Follow-Up.

PURPOSE: Pulp and nail atrophy and asymmetry are commonly seen in thumb duplication. In hypoplasia of both digits, conventional reconstruction or Bilhaut-Cloquet procedure and its modifications may not be possible or may lead to a poor cosmetic outcome. The purpose of the study was to review a reconstruction technique with a neurovascular island flap developed to improve the aesthetic and functional results of treatment. METHODS: Fourteen patients with thumb duplication aged 8 to 18 months were operated between 2002 and 2013 in our center. All patients had significant hypoplasia and asymmetry of the pulp and nail of the digit planned to be retained. A neurovascular island flap including part of the pulp tissue, nail bed, with or without the associated phalangeal bone, was raised from the planned ablated digit base on its single neurovascular bundle. The nail bed, nail fold, and pulp tissue from the 2 digits were apposed with fine sutures under magnification. All patients were followed to monitor the aesthetic, functional, and radiological outcome. RESULTS: The mean follow-up period was 7 years, 11 months. Thirteen patients underwent the flap procedure and all flaps survived. In 1 patient, the flap procedure was aborted because the vascular pedicle was not well formed. The nail width and pulp circumference were restored to a similar size as the contralateral thumb. CONCLUSIONS: In selected cases of thumb duplication with significant pulp hypoplasia and nail asymmetry, the neurovascular island flap is a safe and effective means to restore size and symmetry. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

A process engineering approach to increase organoid yield.

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods, which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence, focusing on measurable parameters of hindgut spheroids, the intermediate step between definitive endoderm and mature organoids. We found that 13% of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number, diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation, we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 µm and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids, respectively, thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids.

Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Dynamic filopodial forces induce accumulation, damage, and plastic remodeling of 3D extracellular matrices.

The mechanical properties of the extracellular matrix (ECM)-a complex, 3D, fibrillar scaffold of cells in physiological environments-modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local microenvironments. Here, we show how cell-generated contractile forces produce substantial irreversible changes to the density and architecture of physiologically relevant ECMs-collagen I and fibrin-in a matter of minutes. We measure the 3D deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these ECM measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale simulations that account for viscoplastic and damage features. Our findings further confirm that plasticity, as a mechanical law to capture remodeling in these networks, is fundamentally tied to material damage via force-driven unbinding of fiber crosslinks. These results characterize in a multiscale manner the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems.

Indoor nanoscale particulate matter-induced coagulation abnormality based on a human 3D microvascular model on a microfluidic chip.

BACKGROUND: A growing body of evidence shows that indoor concentrations of airborne particles are often higher than is typically encountered outdoors. Since exposure to indoor PM2.5 is thought to be associated with cardiovascular disease, the health impacts of indoor air pollution need to be explored. Based on animal models, ambient particulate matter has been proved to promote coagulation which is very likely involved in the pathogenic development of cardiovascular disease. However, animal models are insufficient to predict what will happen with any certainty in humans. For this reason, the precise pathogenic mechanisms behind the development of cardiovascular disease in humans have not yet been determined. RESULTS: We generated a 3D functional human microvascular network in a microfluidic device. This model enables human vascular endothelial cells to form tissue-like microvessels that behave very similarly to human blood vessels. The perfusable microvasculature allows the delivery of particles introduced into these generated human-like microvessels to follow the fluid flow. This exposure path effectively simulates the dynamic movement of airborne nanoscale particles (ANPs) within human vessels. In this study, we first identified the existence of ANPs in indoor air pollution. We then showed that ANPs could activate endothelial cells via ROS induced inflammation, and further resulted in abnormal expression of the coagulation factors (TF, TM and t-PA) involved in coagulation cascades. In addition, we found that a protein could cover ANPs, and this biointeraction could interfere with heparan sulfate (HS). Human organotypic 3D microvessel models provide a bridge for how research outcomes can translate to humans. CONCLUSIONS: The 3D human microvessel model was used to determine the physiological responses of human vessels to ANP stimulation. Based on the obtained data, we concluded that ANPs not only disrupts normal coagulation functions, but also act directly on anticoagulant factors in human vessels. These experimental observations provide a potential biological explanation for the epidemiologically established link between ANPs and coagulation abnormality. This organ-on-chip model may provide a bridge from in vitro results to human responses.

Top-Ten Tips for Imaging the Triangular Fibrocartilaginous Complex.

The triangular fibrocartilage complex (TFCC) is a crucial structure for both maintaining the stability of the distal radioulnar joint (DRUJ) and acting as a cushion for axial loading of the ulnocarpal joint. Injury to the TFCC can lead to early degeneration of the DRUJ and ulnocarpal joint, with resultant chronic wrist pain and weakness. The TFCC is a moderately complex structure with several attachments to the adjacent bony and cartilaginous structures. Familiarity with the anatomy of the TFCC is a prerequisite for identification of TFCC tears. Several pitfalls can occur while assessing the TFCC on magnetic resonance imaging (MRI) if one is not familiar with the MRI appearances. This article illustrates key tips for diagnosing TFCC tears on MRI.

Integrated Analysis of Intracellular Dynamics of MenaINV Cancer Cells in a 3D Matrix.

The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data. We further perform microrheology experiments, integrated with Brownian dynamics simulations of the active cytoskeleton, on metastatic breast cancer cells embedded in a three-dimensional collagen matrix with and without the presence of epidermal growth factor to probe the intracellular mechanical response in a physiologically mimicking scenario. Our results demonstrate that EGF stimulation can alter intracellular stiffness and power output from molecular motor-driven fluctuations in cells overexpressing an invasive isoform of the actin-associated protein Mena.