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Here, we demonstrate an in situ electrostatic actuator that can operate underwater across a wide range of displacements and frequencies, achieving a displacement of approximately 10-μm at 500-Hz and 1-μm at 5-kHz; this performance surpasses that of existing underwater physical actuators. To attain these large displacements at such high speeds, we optimized critical design parameters using a computationally efficient description of the physics of low quality (Q) factor underwater electrostatic actuators. Our theoretical model accurately predicts actuator motion profiles as well as limits of bandwidth and displacement.

Cooking is a tangible, familiar, and delicious tool for teaching physics, which is easy to implement in a university setting. Through our courses at Harvard and UCLA, each year we are engaging hundreds of undergraduate students, primarily non-science majors, in science concepts and the scientific research process. We find that weekly lectures by chefs and professors, paired with edible lab experiments, generate enthusiasm and provide strong motivation for students to learn physics. By the end of the course, students are able to conduct independent scientific research and present their results in a final science fair. Given the considerable broad appeal of food and cooking, the topic could be adapted to other post-secondary as well as secondary-level courses.

There is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that both is palatable and has reduced environmental impact. However, strategies to support the production of cultured meats at the scale required for food consumption will be critical. In this review, we discuss the current challenges and opportunities of using edible scaffolds for scaling up the production of cultured meat. We provide an overview of different types of edible scaffolds, scaffold fabrication techniques, and common scaffold materials. Finally, we highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat.

Tuning of genome structure and function is accomplished by chromatin-binding proteins, which determine the transcriptome and phenotype of the cell. Here we investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that histone H1.0, which compacts nucleosomes into higher-order chromatin fibers, controls genome organization and cellular stress response. We show that histone H1.0 has privileged expression in fibroblasts across tissue types and that its expression is necessary and sufficient to induce myofibroblast activation. Depletion of histone H1.0 prevents cytokine-induced fibroblast contraction, proliferation and migration via inhibition of a transcriptome comprising extracellular matrix, cytoskeletal and contractile genes, through a process that involves locus-specific H3K27 acetylation. Transient depletion of histone H1.0 in vivo prevents fibrosis in cardiac muscle. These findings identify an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling force generation, nuclear organization and gene transcription., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2024.)

Epithelial mechanics and mechanobiology have become 2 important research fields in life sciences and bioengineering. These fields investigate how physical factors induced by cell adhesion and collective behaviors can directly regulate biologic processes, such as organ development and disease progression. Cell mechanics and mechanobiology thus make exciting biophysics education topics to illustrate how fundamental physics principles play a role in regulating cell biology. However, the field currently lacks hands-on activities that engage students in learning science and outreach programs in these topics. One such area is the development of robust hands-on modules that allow students to observe features of cell shape and mechanics and connect them to fundamental physics principles. Here, we demonstrate a workflow that engages students in studying epithelial cell mechanics by using commercial histology slides of frog skin. We show that by using recently developed artificial intelligence-based image-segmentation tools, students can easily quantify different cell morphologic features in a high-throughput manner. Using our workflow, students can reproduce 2 essential findings in cell mechanics: the common gamma distribution of normalized cell aspect ratio in jammed epithelia and the constant ratio between the nuclear and cellular area. Importantly, because the only required instrument for this active learning module is a readily available light microscope and a computer, our module is relatively low cost, as well as portable. These features make the module scalable for students at various education levels and outreach programs. This highly accessible education module provides a fun and engaging way to introduce students to the world of epithelial tissue mechanics.

The integration of intramuscular fat-or marbling-into cultured meat will be critical for meat texture, mouthfeel, flavor, and thus consumer appeal. However, culturing muscle tissue with marbling is challenging since myocytes and adipocytes have different media and scaffold requirements for optimal growth and differentiation. Here, we present an approach to engineer multicomponent tissue using myogenic and adipogenic microtissues. The key innovation in our approach is the engineering of myogenic and adipogenic microtissues using scaffolds with customized physical properties; we use these microtissues as building blocks that spontaneously adhere to produce multicomponent tissue, or marbled cultured meat. Myocytes are grown and differentiated on gelatin nanofiber scaffolds with aligned topology that mimic the aligned structure of skeletal muscle and promotes the formation of myotubes in both primary rabbit skeletal muscle and murine C2C12 cells. Pre-adipocytes are cultured and differentiated on edible gelatin microbead scaffolds, which are customized to have a physiologically-relevant stiffness, and promote lipid accumulation in both primary rabbit and murine 3T3-L1 pre-adipocytes. After harvesting and stacking the individual myogenic and adipogenic microtissues, we find that the resultant multicomponent tissues adhere into intact structures within 6-12 h in culture. The resultant multicomponent 3D tissue constructs show behavior of a solid material with a Young's modulus of ∼ 2 ± 0.4 kPa and an ultimate tensile strength of ∼ 23 ± 7 kPa without the use of additional crosslinkers. Using this approach, we generate marbled cultured meat with ∼ mm to ∼ cm thickness, which has a protein content of ∼ 4 ± 2 g/100 g that is comparable to a conventionally produced Wagyu steak with a protein content of ∼ 9 ± 4 g/100 g. We show the translatability of this layer-by-layer assembly approach for microtissues across primary rabbit cells, murine cell lines, as well as for gelatin and plant-based scaffolds, which demonstrates a strategy to generate edible marbled meats derived from different species and scaffold materials., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro- and nano-topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The recurrence of cancer following chemotherapy treatment is a major cause of death across solid and hematologic cancers. In B-cell acute lymphoblastic leukemia (B-ALL), relapse after initial chemotherapy treatment leads to poor patient outcomes. Here we test the hypothesis that chemotherapy-treated versus control B-ALL cells can be characterized based on cellular physical phenotypes. To quantify physical phenotypes of chemotherapy-treated leukemia cells, we use cells derived from B-ALL patients that are treated for 7 days with a standard multidrug chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VDL). We conduct physical phenotyping of VDL-treated versus control cells by tracking the sequential deformations of single cells as they flow through a series of micron-scale constrictions in a microfluidic device; we call this method Quantitative Cyclical Deformability Cytometry. Using automated image analysis, we extract time-dependent features of deforming cells including cell size and transit time (TT) with single-cell resolution. Our findings show that VDL-treated B-ALL cells have faster TTs and transit velocity than control cells, indicating that VDL-treated cells are more deformable. We then test how effectively physical phenotypes can predict the presence of VDL-treated cells in mixed populations of VDL-treated and control cells using machine learning approaches. We find that TT measurements across a series of sequential constrictions can enhance the classification accuracy of VDL-treated cells in mixed populations using a variety of classifiers. Our findings suggest the predictive power of cell physical phenotyping as a complementary prognostic tool to detect the presence of cells that survive chemotherapy treatment. Ultimately such complementary physical phenotyping approaches could guide treatment strategies and therapeutic interventions. Insight box Cancer cells that survive chemotherapy treatment are major contributors to patient relapse, but the ability to predict recurrence remains a challenge. Here we investigate the physical properties of leukemia cells that survive treatment with chemotherapy drugs by deforming individual cells through a series of micron-scale constrictions in a microfluidic channel. Our findings reveal that leukemia cells that survive chemotherapy treatment are more deformable than control cells. We further show that machine learning algorithms applied to physical phenotyping data can predict the presence of cells that survive chemotherapy treatment in a mixed population. Such an integrated approach using physical phenotyping and machine learning could be valuable to guide patient treatments., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

PODXL, a protein that is dysregulated in multiple cancers, plays an important role in promoting cancer metastasis. In this study, we report that RNA editing promotes the inclusion of a PODXL alternative exon. The resulting edited PODXL long isoform is more prone to protease digestion and has the strongest effects on reducing cell migration and cisplatin chemoresistance among the three PODXL isoforms (short, unedited long, and edited long isoforms). Importantly, the editing level of the PODXL recoding site and the inclusion level of the PODXL alternative exon are strongly associated with overall patient survival in Kidney Renal Clear Cell Carcinoma (KIRC). Supported by significant enrichment of exonic RNA editing sites in alternatively spliced exons, we hypothesize that exonic RNA editing sites may enhance proteomic diversity through alternative splicing, in addition to amino acid changes, a previously under-appreciated aspect of RNA editing function., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Cultured meat has potential to diversify methods for protein production, but innovations in production efficiency will be required to make cultured meat a feasible protein alternative. Microcarriers provide a strategy to culture sufficient volumes of adherent cells in a bioreactor that are required for meat products. However, cell culture on inedible microcarriers involves extra downstream processing to dissociate cells prior to consumption. Here, we present edible microcarriers that can support the expansion and differentiation of myogenic cells in a single bioreactor system. To fabricate edible microcarriers with a scalable process, we used water-in-oil emulsions as templates for gelatin microparticles. We also developed a novel embossing technique to imprint edible microcarriers with grooved topology in order to test if microcarriers with striated surface texture can promote myoblast proliferation and differentiation in suspension culture. In this proof-of-concept demonstration, we showed that edible microcarriers with both smooth and grooved surface topologies supported the proliferation and differentiation of mouse myogenic C2C12 cells in a suspension culture. The grooved edible microcarriers showed a modest increase in the proliferation and alignment of myogenic cells compared to cells cultured on smooth, spherical microcarriers. During the expansion phase, we also observed the formation of cell-microcarrier aggregates or 'microtissues' for cells cultured on both smooth and grooved microcarriers. Myogenic microtissues cultured with smooth and grooved microcarriers showed similar characteristics in terms of myotube length, myotube volume fraction, and expression of myogenic markers. To establish feasibility of edible microcarriers for cultured meat, we showed that edible microcarriers supported the production of myogenic microtissue from C2C12 or bovine satellite muscle cells, which we harvested by centrifugation into a cookable meat patty that maintained its shape and exhibited browning during cooking. These findings demonstrate the potential of edible microcarriers for the scalable production of cultured meat in a single bioreactor., (Copyright © 2022. Published by Elsevier Ltd.)

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The study of physical and mechanical features of cancer cells, or cancer cell mechanobiology, is a new frontier in cancer research. Such studies may enhance our understanding of the disease process, especially mechanisms associated with cancer cell invasion and metastasis, and may help the effort of developing diagnostic biomarkers and therapeutic drug targets. Cancer cell mechanobiological changes are associated with the complex interplay of activation/inactivation of multiple signaling pathways, which can occur at both the genetic and epigenetic levels, and the interactions with the cancer microenvironment. It has been shown that metastatic tumor cells are more compliant than morphologically similar benign cells in actual human samples. Subsequent studies from us and others further demonstrated that cell mechanical properties are strongly associated with cancer cell invasive and metastatic potential, and thus may serve as a diagnostic marker of detecting cancer cells in human body fluid samples. In this review, we provide a brief narrative of the molecular mechanisms underlying cancer cell mechanobiology, the technological platforms utilized to study cancer cell mechanobiology, the status of cancer cell mechanobiological studies in various cancer types, and the potential clinical applications of cancer cell mechanobiological study in cancer early detection, diagnosis, and treatment., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 Chinese National Cancer Center. Published by Elsevier B.V.)

The mutant nuclear lamin protein (progerin) produced in Hutchinson-Gilford progeria syndrome (HGPS) results in loss of arterial smooth muscle cells (SMCs), but the mechanism has been unclear. We found that progerin induces repetitive nuclear membrane (NM) ruptures, DNA damage, and cell death in cultured SMCs. Reducing lamin B1 expression and exposing cells to mechanical stress - to mirror conditions in the aorta - triggered more frequent NM ruptures. Increasing lamin B1 protein levels had the opposite effect, reducing NM ruptures and improving cell survival. Remarkably, raising lamin B1 levels increased nuclear compliance in cells and was able to offset the increased nuclear stiffness caused by progerin. In mice, lamin B1 expression in aortic SMCs is normally very low, and in mice with a targeted HGPS mutation (LmnaG609G), levels of lamin B1 decrease further with age while progerin levels increase. Those observations suggest that NM ruptures might occur in aortic SMCs in vivo. Indeed, studies in LmnaG609G mice identified NM ruptures in aortic SMCs, along with ultrastructural abnormalities in the cell nucleus that preceded SMC loss. Our studies identify NM ruptures in SMCs as likely causes of vascular pathology in HGPS.

The goal of this viewpoint is to promote an integrated and holistic framework for food literacy on college campuses. We propose that a framework to promote an effective understanding of food should encompass social, political, scientific, and personal dimensions; integrating these elements into university curricula and campus culture can empower students to become more engaged food citizens, with implications for their own food choices, and also for the broader food system. Emerging findings show that curricular interventions designed to educate about food system-environment connections can motivate students to reduce red meat and increase vegetable consumption. This viewpoint also lays the foundation for future studies to quantify the impact of increased knowledge on food choices, which can ultimately impact the health and wellbeing of both people and the planet.

Immune cells can sense and respond to biophysical cues - from dynamic forces to spatial features - during their development, activation, differentiation and expansion. These biophysical signals regulate a variety of immune cell functions such as leukocyte extravasation, macrophage polarization, T cell selection and T cell activation. Recent studies have advanced our understanding on immune responses to biophysical cues and the underlying mechanisms of mechanotransduction, which provides rational basis for the design and development of immune-modulatory therapeutics. This review discusses the recent progress in mechanosensing and mechanotransduction of immune cells, particularly monocytes/macrophages and T lymphocytes, and features new biomaterial designs and biomedical devices that translate these findings into biomedical applications., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer cell mechanotype changes are newly recognized cancer phenotypic events, whereas metastatic cancer cells show decreased cell stiffness and increased deformability relative to normal cells. To further examine how cell mechanotype changes in early stages of cancer transformation and progression, an in vitro multi-step human urothelial cell carcinogenic model was used to measure cellular Young's modulus, deformability, and transit time using single-cell atomic force microscopy, microfluidic-based deformability cytometry, and quantitative deformability cytometry, respectively. Measurable cell mechanotype changes of stiffness, deformability, and cell transit time occur early in the transformation process. As cells progress from normal, to preinvasive, to invasive cells, Young's modulus of stiffness decreases and deformability increases gradually. These changes were confirmed in three-dimensional cultured microtumor masses and urine exfoliated cells directly from patients. Using gene screening and proteomics approaches, we found that the main molecular pathway implicated in cell mechanotype changes appears to be epithelial to mesenchymal transition., (Copyright © 2020 Yu, Lu, Sharma, Ly, Di Carlo, Rowat, LeClaire, Kim, Chow, Gimzewski and Rao.)

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner., Competing Interests: Declaration of Interests The authors declare no competing interests. Based on this work, patent no: 63/002,828 “Compositions and methods for treating dysregulated wound healing” has been filed and assigned to the Regents of the University of California., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought., Competing Interests: Declaration of Interests M.D.H. is president, co-founder, and shareholder of SynderBio, Inc., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Evolving norms and complex food environments may require new skill sets and mind-sets to maintain a healthy diet. Food literacy acknowledges the influence of external factors in shaping a person's development and application of the knowledge, skills, and behaviors required for healthy eating. Food literacy among college students is not well understood; however, higher education presents a unique opportunity for promoting food literacy., Objective: This study aimed to identify a range of challenges, opportunities, and motivators for students to develop and apply food literacy in a university setting., Design: Eleven focus groups were conducted with four student subpopulations: three with residential undergraduates, three with off-campus undergraduates, three with graduate students, and two with students using food security resources., Participants/setting: Eighty-two students enrolled at a large, diverse, public university in an urban setting in California., Analysis: Guided by an ecological perspective, transcripts were analyzed using an integrated approach. This involved an inductive development of themes and deductive organization of themes according to research aims., Results: We developed a novel model as a starting point for understanding and addressing the dynamic challenges, opportunities, and motivators for students to develop and apply food literacy. Challenges include the physical food environment, confusing information, capacity and resource constraints, and social tensions. Opportunities include media and the Internet, academic courses, peer learning, campus resources, and dining halls. Motivators include health, social responsibility, personal development, and enjoyment and bonding., Conclusions: Students view college as an appropriate time to develop food literacy and the university as a trusted partner. However, efforts to promote food literacy should acknowledge perceived challenges and varying motivations for engaging with food., (Copyright © 2020 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

Fallopian tube secretory epithelial cells (FTSECs) are likely the main precursor cell type of high-grade serous ovarian cancers (HGSOCs), but these tumors may also arise from ovarian surface epithelial cells (OSECs). We profiled global landscapes of gene expression and active chromatin to characterize molecular similarities between OSECs (n = 114), FTSECs (n = 74), and HGSOCs (n = 394). A one-class machine learning algorithm predicts that most HGSOCs derive from FTSECs, with particularly high FTSEC scores in mesenchymal-type HGSOCs (p adj  < 8 × 10 -4 ). However, a subset of HGSOCs likely derive from OSECs, particularly HGSOCs of the proliferative type (p adj  < 2 × 10 -4 ), suggesting a dualistic model for HGSOC origins. Super-enhancer (SE) landscapes were also more similar between FTSECs and HGSOCs than between OSECs and HGSOCs (p < 2.2 × 10 -16 ). The SOX18 transcription factor (TF) coincided with a HGSOC-specific SE, and ectopic overexpression of SOX18 in FTSECs caused epithelial-to-mesenchymal transition, indicating that SOX18 plays a role in establishing the mesenchymal signature of fallopian-derived HGSOCs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Metastasis is a fundamentally physical process in which cells deform through narrow gaps and generate forces to invade surrounding tissues. While it is commonly thought that increased cell deformability is an advantage for invading cells, we previously found that more invasive pancreatic ductal adenocarcinoma (PDAC) cells are stiffer than less invasive PDAC cells. Here we investigate potential mechanisms of the simultaneous increase in PDAC cell stiffness and invasion, focusing on the contributions of myosin II, Arp2/3, and formins., Method: We measure cell invasion using a 3D scratch wound invasion assay and cell stiffness using atomic force microscopy (AFM). To determine the effects of actin- and myosin-mediated force generation on cell stiffness and invasion, we treat cells with pharmacologic inhibitors of myosin II (blebbistatin), Arp2/3 (CK-666), and formins (SMIFH2)., Results: We find that the activity of myosin II, Arp2/3, and formins all contribute to the stiffness of PDAC cells. Interestingly, we find that the invasion of PDAC cell lines is differentially affected when the activity of myosin II, Arp2/3, or formins is inhibited, suggesting that despite having similar tissue origins, different PDAC cell lines may rely on different mechanisms for invasion., Conclusions: These findings deepen our knowledge of the factors that regulate cancer cell mechanotype and invasion, and incite further studies to develop therapeutics that target multiple mechanisms of invasion for improved clinical benefit., (© Biomedical Engineering Society 2019.)

The mechanical properties of a cell, which include parameters such as elasticity, inner pressure, and tensile strength, are extremely important because changes in these properties are indicative of diseases ranging from diabetes to malignant transformation. Considering the heterogeneity within a population of cancer cells, a robust measurement system at the single cell level is required for research and in clinical purposes. In this study, a potential microfluidic device for high-throughput and practical mechanotyping were developed to investigate the deformability and sizes of cells through a single run. This mechanotyping device consisted of two different sizes of consecutive constrictions in a microchannel and measured the size of cells and related deformability during transit. Cell deformability was evaluated based on the transit and on the effects of cytoskeleton-affecting drugs, which were detected within 50 ms. The mechanotyping device was able to also measure a cell cycle without the use of fluorescent or protein tags.

Background: Over 96% of high-grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63, which shares many of the downstream targets and functions of p53, is rarely mutated in cancer., Methods: A novel strategy is presented for circumventing alterations in p53 by inducing the tumor-suppressor isoform TAp63 (transactivation domain of tumor protein p63) through its direct downstream target, microRNA-130b (miR-130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer., Results: Treatment with miR-130b resulted in: 1) decreased migration/invasion in HEYA8 cells (p53 wild-type) and disruption of multicellular spheroids in OVCAR8 cells (p53-mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B-cell lymphoma (Bcl)-inhibitor B-cell lymphoma 2-like protein 11 (BIM). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR-130b. In vivo, combined miR-130b plus CDDP exhibited greater therapeutic efficacy than miR-130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR-130b in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP-treated mice, and 20% of DOPC-miR-130b plus CDDP-treated mice were living tumor free. Systemic injections of scL-miR-130b plus CDDP in a clinically tested, tumor-targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts., Conclusions: The miR-130b/TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad-spectrum therapeutic options for the majority of p53-altered cancers., (© 2019 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.)

DYT1 dystonia is a neurological movement disorder that is caused by a loss-of-function mutation in the DYT1 / TOR1A gene, which encodes torsinA, a conserved luminal ATPases-associated with various cellular activities (AAA+) protein. TorsinA is required for the assembly of functional linker of nucleoskeleton and cytoskeleton (LINC) complexes, and consequently the mechanical integration of the nucleus and the cytoskeleton. Despite the potential implications of altered mechanobiology in dystonia pathogenesis, the role of torsinA in regulating cellular mechanical phenotype, or mechanotype, in DYT1 dystonia remains unknown. Here, we define the deformability of mouse fibroblasts lacking functional torsinA as well as human fibroblasts isolated from DYT1 dystonia patients. We find that the deletion of torsinA or the expression of torsinA containing the DYT1 dystonia-causing ΔE302/303 (ΔE) mutation results in more deformable cells. We observe a similar increased deformability of mouse fibroblasts that lack lamina-associated polypeptide 1 (LAP1), which interacts with and stimulates the ATPase activity of torsinA in vitro , as well as with the absence of the LINC complex proteins, Sad1/UNC-84 1 (SUN1) and SUN2, lamin A/C, or lamin B1. Consistent with these findings, we also determine that DYT1 dystonia patient-derived fibroblasts are more compliant than fibroblasts isolated from unafflicted individuals. DYT1 dystonia patient-derived fibroblasts also exhibit increased nuclear strain and decreased viability following mechanical stretch. Taken together, our results establish the foundation for future mechanistic studies of the role of cellular mechanotype and LINC-dependent nuclear-cytoskeletal coupling in regulating cell survival following exposure to mechanical stresses.

Critical functions of immune cells require them to rapidly change their shape and generate forces in response to cues from their surrounding environment. However, little is known about how soluble factors that may be present in the microenvironment modulate key aspects of cellular mechanobiology-such as immune cell deformability and force generation-to impact functions such as phagocytosis and migration. Here we show that signaling by soluble stress hormones through β-adrenoceptors (β-AR) reduces the deformability of macrophages; this is dependent on changes in the organization of the actin cytoskeleton and is associated with functional changes in phagocytosis and migration. Pharmacologic interventions reveal that the impact of β-AR signaling on macrophage deformability is dependent on actin-related proteins 2/3, indicating that stress hormone signaling through β-AR shifts actin organization to favor branched structures rather than linear unbranched actin filaments. These findings show that through remodeling of the actin cytoskeleton, β-AR-mediated stress hormone signaling modulates macrophage mechanotype to impact functions that play a critical role in immune response.-Kim, T.-H., Ly, C., Christodoulides, A., Nowell, C. J., Gunning, P. W., Sloan, E. K., Rowat, A. C. Stress hormone signaling through β-adrenergic receptors regulates macrophage mechanotype and function.

Cell deformability is a label-free biomarker of cell state in physiological and disease contexts ranging from stem cell differentiation to cancer progression. Harnessing deformability as a phenotype for screening applications requires a method that can simultaneously measure the deformability of hundreds of cell samples and can interface with existing high throughput facilities. Here we present a scalable cell filtration device, which relies on the pressure-driven deformation of cells through a series of pillars that are separated by micron-scale gaps on the timescale of seconds: less deformable cells occlude the gaps more readily than more deformable cells, resulting in decreased filtrate volume which is measured using a plate reader. The key innovation in this method is that we design customized arrays of individual filtration devices in a standard 96-well format using soft lithography, which enables multiwell input samples and filtrate outputs to be processed with higher throughput using automated pipette arrays and plate readers. To validate high throughput filtration to detect changes in cell deformability, we show the differential filtration of human ovarian cancer cells that have acquired cisplatin-resistance, which is corroborated with cell stiffness measurements using quantitative deformability cytometry. We also demonstrate differences in the filtration of human cancer cell lines, including ovarian cancer cells that overexpress transcription factors (Snail, Slug), which are implicated in epithelial-to-mesenchymal transition; breast cancer cells (malignant versus benign); and prostate cancer cells (highly versus weekly metastatic). We additionally show how the filtration of ovarian cancer cells is affected by treatment with drugs known to perturb the cytoskeleton and the nucleus. Our results across multiple cancer cell types with both genetic and pharmacologic manipulations demonstrate the potential of this scalable filtration device to screen cells based on their deformability.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane-associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis. Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg Cancer Res; 78(21); 6086-97. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The physical properties of cells are promising biomarkers for cancer diagnosis and prognosis. Here we determine the physical phenotypes that best distinguish human cancer cell lines, and their relationship to cell invasion. We use the high throughput, single-cell microfluidic method, quantitative deformability cytometry (q-DC), to measure six physical phenotypes including elastic modulus, cell fluidity, transit time, entry time, cell size, and maximum strain at rates of 102 cells per second. By training a k-nearest neighbor machine learning algorithm, we demonstrate that multiparameter analysis of physical phenotypes enhances the accuracy of classifying cancer cell lines compared to single parameters alone. We also discover a set of four physical phenotypes that predict invasion; using these four parameters, we generate the physical phenotype model of invasion by training a multiple linear regression model with experimental data from a set of human ovarian cancer cells that overexpress a panel of tumor suppressor microRNAs. We validate the model by predicting invasion based on measured physical phenotypes of breast and ovarian human cancer cell lines that are subject to genetic or pharmacologic perturbations. Taken together, our results highlight how physical phenotypes of single cells provide a biomarker to predict the invasion of cancer cells.

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, E a , and power-law exponent, β. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of E a  = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (E a  = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that E a increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that E a increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Wrinkling of thin films and membranes can occur due to various mechanisms such as growth and/or mismatch between the mechanical properties of the film and substrate. However, the physical origins of dynamic wrinkling in soft membranes are still not fully understood. Here we use milk skin as a tractable experimental system to investigate the physics of wrinkle formation in a thin, poroelastic film. Upon heating milk, a micron-thick hydrogel of denatured proteins and fat globules forms at the air-water interface. Over time, we observe an increase in the total length of wrinkles. By confocal imaging and profilometry, we determine that the composition and thickness of the milk skin appears to be homogeneous over the length scale of the wrinkles, excluding differences in milk skin composition as a major contributor to wrinkling. To explain the physical origins of wrinkle growth, we describe theory that considers the milk skin as a thin, poroelastic film where pressure is generated by the evaporative-driven flow of solvent across the film; this imparts in-plane stresses in the milk skin, which cause wrinkling. Viscous effects can explain the time-dependent growth of wrinkles. Our theoretical predictions of the effects of relative humidity on the total length of wrinkles over time are consistent with our experimental results. Our findings provide insight into the physics of the common phenomenon of milk skin wrinkling, and identify hydration gradients as another physical mechanism that can drive morphological instabilities in soft matter.

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. β-adrenergic receptor (βAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of β-adrenergic signaling by βAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that βAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that βAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological β-blockade or genetic knockout of the β 2 -adrenergic receptor. Our results identify a β 2 -adrenergic-Ca 2+ -actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Young's modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Young's modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

The activities of pathways that regulate malignant transformation can be influenced by microRNAs (miRs). Recently, we showed that increased expression of five tumour-suppressor miRs, miR-508-3p, miR-508-5p, miR-509-3p, miR-509-5p and miR-130b-3p, correlate with improved clinical outcomes in human ovarian cancer patients, and that miR-509-3p attenuates invasion of ovarian cancer cell lines. Here, we investigate the mechanism underlying this reduced invasive potential by assessing the impact of these five miRs on the physical properties of cells. Human ovarian cancer cells (HEYA8, OVCAR8) that are transfected with miR mimics representing these five miRs exhibit decreased invasion through collagen matrices, increased cell size and reduced deformability as measured by microfiltration and microfluidic assays. To understand the molecular basis of altered invasion and deformability induced by these miRs, we use predicted and validated mRNA targets that encode structural and signalling proteins that regulate cell mechanical properties. Combined with analysis of gene transcripts by real-time PCR and image analysis of F-actin in single cells, our results suggest that these tumour-suppressor miRs may alter cell physical properties by regulating the actin cytoskeleton. Our findings provide biophysical insights into how tumour-suppressor miRs can regulate the invasive behaviour of ovarian cancer cells, and identify potential therapeutic targets that may be implicated in ovarian cancer progression., (© 2016 The Authors.)

The mechanical phenotype or 'mechanotype' of cells is emerging as a potential biomarker for cell types ranging from pluripotent stem cells to cancer cells. Using a microfluidic device, cell mechanotype can be rapidly analyzed by measuring the time required for cells to deform as they flow through constricted channels. While cells typically exhibit deformation timescales, or transit times, on the order of milliseconds to tens of seconds, transit times can span several orders of magnitude and vary from day to day within a population of single cells; this makes it challenging to characterize different cell samples based on transit time data. Here we investigate how variability in transit time measurements depends on both experimental factors and heterogeneity in physical properties across a population of single cells. We find that simultaneous transit events that occur across neighboring constrictions can alter transit time, but only significantly when more than 65% of channels in the parallel array are occluded. Variability in transit time measurements is also affected by the age of the device following plasma treatment, which could be attributed to changes in channel surface properties. We additionally investigate the role of variability in cell physical properties. Transit time depends on cell size; by binning transit time data for cells of similar diameters, we reduce measurement variability by 20%. To gain further insight into the effects of cell-to-cell differences in physical properties, we fabricate a panel of gel particles and oil droplets with tunable mechanical properties. We demonstrate that particles with homogeneous composition exhibit a marked reduction in transit time variability, suggesting that the width of transit time distributions reflects the degree of heterogeneity in subcellular structure and mechanical properties within a cell population. Our results also provide fundamental insight into the physical underpinnings of transit measurements: transit time depends strongly on particle elastic modulus, and weakly on viscosity and surface tension. Based on our findings, we present a comprehensive methodology for designing, analyzing, and reducing variability in transit time measurements; this should facilitate broader implementation of transit experiments for rapid mechanical phenotyping in basic research and clinical settings.

Despite recent progress in cancer research, the exact nature of malignant transformation and its progression is still not fully understood. Particularly metastasis, which accounts for most cancer death, is a very complex process, and new treatment strategies require a more comprehensive understanding of underlying regulatory mechanisms. Recently, the sympathetic nervous system (SNS) has been implicated in cancer progression and beta-blockers have been identified as a novel strategy to limit metastasis. This review discusses evidence that SNS signaling regulates metastasis by modulating the physical characteristics of tumor cells, tumor-associated immune cells and the extracellular matrix (ECM). Altered mechanotype is an emerging hallmark of cancer cells that is linked to invasive phenotype and treatment resistance. Mechanotype also influences crosstalk between tumor cells and their environment, and may thus have a critical role in cancer progression. First, we discuss how neural signaling regulates metastasis and how SNS signaling regulates both biochemical and mechanical properties of tumor cells, immune cells and the ECM. We then review our current knowledge of the mechanobiology of cancer with a focus on metastasis. Next, we discuss links between SNS activity and tumor-associated inflammation, the mechanical properties of immune cells, and how the physical properties of the ECM regulate cancer and metastasis. Finally, we discuss the potential for clinical translation of our knowledge of cancer mechanobiology to improve diagnosis and treatment.

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E-3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E-3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3p's most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers., Competing Interests: The authors declare no conflicts of interests.

Cell mechanical phenotype or 'mechanotype' is emerging as a valuable label-free biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRas(V12)-transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application.

Diffusion is critical to physiological processes ranging from gas exchange across alveoli to transport within individual cells. In the classroom, however, it can be challenging to convey the concept of diffusion on the microscopic scale. In this article, we present a series of three exercises that use food and cooking to illustrate diffusion theory and Fick's first law. These exercises are part of a 10-wk undergraduate course that uses food and cooking to teach fundamental concepts in physiology and biophysics to students, including nonscience majors. Consistent demonstration of practical applications in a classroom setting has the potential to fundamentally change how students view the role of science in their lives (15)., (Copyright © 2015 The American Physiological Society.)

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~10(2) cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We study elasticity-driven morphological transitions of soft spherical core-shell structures in which the core can be treated as an isotropic elastic continuum and the surface or shell as a tensionless liquid layer, whose elastic response is dominated by bending. To generate the transitions, we consider the case where the surface area of the liquid layer is increased for a fixed amount of interior elastic material. We find that generically there is a critical excess surface area at which the isotropic sphere becomes unstable to buckling. At this point it adopts a lower symmetry wrinkled structure that can be described by a spherical harmonic deformation. We study the dependence of the buckled sphere and critical excess area of the transition on the elastic parameters and size of the system. We also relate our results to recent experiments on the wrinkling of gel-filled vesicles as their interior volume is reduced. The theory may have broader applications to a variety of related structures from the macroscopic to the microscopic, including the wrinkling of dried peas, raisins, as well as the cell nucleus.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

A microfluidic technique is described to encapsulate living cells in alginate hydrogel microparticles generated from monodisperse double-emulsion templates. A microcapillary device is used to fabricate double emulsion templates composed of an alginate drop surrounded by a mineral oil shell. Hydrogel formation begins when the alginate drop separates from the mineral oil shell and comes into contact with Ca(2+) ions in the continuous phase. Alginate hydrogel microparticles with diameters ranging from 60 to 230 µm are obtained. 65% of the cells encapsulated in the alginate microparticles were viable after one week. The technique provides a useful means to encapsulate the living cells in monodisperse hydrogel microparticles., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Young's modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.