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  • Electrophysiological properties of neurons grown on soft polymer scaffolds reveal the potential to develop neuromimetic culture environments
    Integr. Biol. (IF 2.774) Pub Date : 2020-01-10
    Evans M, Al-Shakli A, Chari D.

    Tissue engineering methodologies for various physiological systems are seeing a significant trend towards 3D cell culture in or on ‘soft’ polymeric hydrogel materials, widely considered to provide a more biomimetic environment for cell growth versus ‘hard’ materials such as glass or plastic. Progress has been slower with 3D neural cell culture with current studies overwhelmingly reliant on hard substrates. Accordingly, our knowledge of the alterations in electrochemical properties of neurons propagated in soft materials is relatively limited. In this study, primary cortical neurons and glial cells were seeded onto the surface of collagen hydrogels and grown in vitro for 7–8 days. At this time, neurons had formed a complex neurite web interspersed with astrocytes. Neuronal patch clamp recordings revealed voltage-gated Na+ and K+ currents in voltage clamp and action potentials in current clamp. When measured at voltages close to maximum activation, both currents were >1 nA in mean amplitude. When compared to primary cortical neurons cultured on glass coverslips, but otherwise under similar conditions (Evans et al., 2017), the Na+ current from hydrogel neurons was found to be significantly larger although there were no differences in the K+ current amplitude, membrane potential, input resistance or cell capacitance. We speculate that the larger size of the neuronal voltage-dependent Na+ current in the hydrogels is related to the better biomimetic properties of the soft material, being close to values reported for neurons recorded in brain slices. The results highlight the potential benefits offered by neuronal culture on soft and biomimetic polymeric materials for neural tissue engineering studies.

    更新日期:2020-01-10
  • Matrix degradation regulates osteoblast protrusion dynamics and individual migration
    Integr. Biol. (IF 2.774) Pub Date : 2020-01-10
    Movilla N, Valero C, Borau C, et al.

    Protrusions are one of the structures that cells use to sense their surrounding environment in a probing and exploratory manner as well as to communicate with other cells. In particular, osteoblasts embedded within a 3D matrix tend to originate a large number of protrusions compared to other type of cells. In this work, we study the role that mechanochemical properties of the extracellular matrix (ECM) play on the dynamics of these protrusions, namely, the regulation of the size and number of emanating structures. In addition, we also determine how the dynamics of the protrusions may lead the 3D movement of the osteoblasts. Significant differences were found in protrusion size and cell velocity, when degradation activity due to metalloproteases was blocked by means of an artificial broad-spectrum matrix metalloproteinase inhibitor, whereas stiffening of the matrix by introducing transglutaminase crosslinking, only induced slight changes in both protrusion size and cell velocity, suggesting that the ability of cells to create a path through the matrix is more critical than the matrix mechanical properties themselves. To confirm this, we developed a cell migration computational model in 3D including both the mechanical and chemical properties of the ECM as well as the protrusion mechanics, obtaining good agreement with experimental results.

    更新日期:2020-01-10
  • Correlation of mRNA delivery timing and protein expression in lipid-based transfection
    Integr. Biol. (IF 2.774) Pub Date : 2019-12-17
    Reiser A, Woschée D, Mehrotra N, et al.

    Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

    更新日期:2020-01-08
  • Co-culture of functionally enriched cancer stem-like cells and cancer-associated fibroblasts for single-cell whole transcriptome analysis
    Integr. Biol. (IF 2.774) Pub Date : 2019-12-10
    Chen Y, Jung S, Zhang Z, et al.

    Considerable evidence suggests that breast cancer development and metastasis are driven by cancer stem-like cells (CSCs). Due to their unique role in tumor initiation, the interaction between CSCs and stromal cells is especially critical. In this work, we developed a platform to reliably isolate single cells in suspension and grow single-cell-derived spheres for functional enrichment of CSCs. The platform also allows adherent culture of stromal cells for cancer-stromal interaction. As a proof of concept, we grew SUM149 breast cancer cells and successfully formed single-cell-derived spheres. Cancer-associated fibroblasts (CAFs) as stromal cells were found to significantly enhance the formation and growth of cancer spheres, indicating elevated tumor-initiation potential. After on-chip culture for 14 days, we retrieved single-cell derived spheres with and without CAF co-culture for single-cell transcriptome sequencing. Whole transcriptome analysis highlights that CAF co-culture can boost cancer stemness especially ALDHhigh CSCs and alter epithelial/mesenchymal status. Single-cell resolution allows identification of individual CSCs and investigation of cancer cellular heterogeneity. Incorporating whole transcriptome sequencing data with public patient database, we discovered novel genes associated with cancer-CAF interaction and critical to patient survival. The preliminary works demonstrated a reliable platform for enrichment of CSCs and studies of cancer-stromal interaction.

    更新日期:2020-01-08
  • Erratum: Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter
    Integr. Biol. (IF 2.774) Pub Date : 2019-12-02
    Manna P, Hung S, Mukherjee S, et al.

    Integrative Biology Journal

    更新日期:2020-01-08
  • Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flow
    Integr. Biol. (IF 2.774) Pub Date : 2019-12-17
    Kim S, Hammer D.

    In order to perform critical immune functions at sites of inflammation, circulatory T lymphocytes must be able to arrest, adhere, migrate and transmigrate on the endothelial surface. This progression of steps is coordinated by cellular adhesion molecules (CAMs), chemokines, and selectins presented on the endothelium. Two important interactions are between Lymphocyte Function-associated Antigen-1 (LFA-1) and Intracellular Adhesion Molecule-1 (ICAM-1) and also between Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Molecule-1 (VCAM-1). Recent studies have shown that T lymphocytes and other cell types can migrate upstream (against the direction) of flow through the binding of LFA-1 to ICAM-1. Since upstream migration of T cells depends on a specific adhesive pathway, we hypothesized that mechanotransduction is critical to migration, and that signals might allow T-cells to remember their direction of migration after the flow is terminated. Cells on ICAM-1 surfaces migrate against the shear flow, but the upstream migration reverts to random migration after the flow is stopped. Cells on VCAM-1 migrate with the direction of flow. However, on surfaces that combine ICAM-1 and VCAM-1, cells crawl upstream at a shear rate of 800 s−1 and continue migrating in the upstream direction for at least 30 minutes after the flow is terminated—we call this ‘migrational memory’. Post-flow upstream migration on VCAM-1/ICAM-1 surfaces is reversed upon the inhibition of PI3K, but conserved with cdc42 and Arp2/3 inhibitors. Using an antibody against VLA-4, we can block migrational memory on VCAM-1/ICAM-1 surfaces. Using a soluble ligand for VLA-4 (sVCAM-1), we can promote migrational memory on ICAM-1 surfaces. These results indicate that, while upstream migration under flow requires LFA-1 binding to immobilized ICAM-1, signaling from VLA-4 and PI3K activity is required for the migrational memory of CD4+ T cells. These results indicate that crosstalk between integrins potentiates the signal of upstream migration.

    更新日期:2019-12-19
  • Phenotypic chemical and mutant screening of zebrafish larvae using an on-demand response to electric stimulation
    Integr. Biol. (IF 2.774) Pub Date : 2019-12-17
    Khalili A, Peimani A, Safarian N, et al.

    Behavioral responses of zebrafish larvae to environmental cues are important functional readouts that should be evoked on-demand and studied phenotypically in behavioral, genetical and developmental investigations. Very recently, it was shown that zebrafish larvae execute a voluntary and oriented movement toward the positive electrode of an electric field along a microchannel. Phenotypic characterization of this response was not feasible due to larva’s rapid movement along the channel. To overcome this challenge, a microfluidic device was introduced to partially immobilize the larva’s head while leaving its mid-body and tail unrestrained in a chamber to image motor behaviors in response to electric stimulation, hence achieving quantitative phenotyping of the electrically evoked movement in zebrafish larvae. The effect of electric current on the tail-beat frequency and response duration of 5–7 days postfertilization zebrafish larvae was studied. Investigations were also performed on zebrafish exposed to neurotoxin 6-hydroxydopamine and larvae carrying a pannexin1a (panx1a) gene knockout, as a proof of principle applications to demonstrate on-demand movement behavior screening in chemical and mutant assays. We demonstrated for the first time that 6-hydroxydopamine leads to electric response impairment, levodopa treatment rescues the response and panx1a is involved in the electrically evoked movement of zebrafish larvae. We envision that our technique is broadly applicable as a screening tool to quantitatively examine zebrafish larvae’s movements in response to physical and chemical stimulations in investigations of Parkinson’s and other neurodegenerative diseases, and as a tool to combine recent advances in genome engineering of model organisms to uncover the biology of electric response.

    更新日期:2019-12-19
  • Substrate curvature induces fallopian tube epithelial cell invasion via cell–cell tension in a model of ovarian cortical inclusion cysts
    Integr. Biol. (IF 2.774) Pub Date : 2019-11-13
    Fleszar A, Walker A, Kreeger P, et al.

    Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell–cell connections were essential for increased invasion on substrates with higher curvature, while cell–substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases—a cell–cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix.

    更新日期:2019-11-30
  • Evaluating Vascularization of Heterotopic Islet Constructs for Type 1 Diabetes Using an In Vitro Platform
    Integr. Biol. (IF 2.774) Pub Date : 2019-11-13
    Bowles A, Ishahak M, Glover S, et al.

    Type 1 diabetes (T1D) results from the autoimmune destruction of β-cells within the pancreatic islets of Langerhans. Clinical islet transplantation from healthy donors is proposed to ameliorate symptoms, improve quality of life, and enhance the life span of afflicted T1D patients. However, post-transplant outcomes are dependent on the survival of the transplanted islets, which relies on the engraftment of the islets with the recipient’s vasculature among other factors. Treatment strategies to improve engraftment include combining islets with supporting cells including endothelial cells (EC) and mesenchymal stem cells (MSC), dynamic cells capable of robust immunomodulatory and vasculogenic effects. In this study, we developed an in vitro model of transplantation to investigate the cellular mechanisms that enhance rapid vascularization of heterotopic islet constructs. Self-assembled vascular beds of fluorescently stained EC served as reproducible in vitro transplantation sites. Heterotopic islet constructs composed of islets, EC, and MSC were transferred to vascular beds for modeling transplantation. Time-lapsed imaging was performed for analysis of the vascular bed remodeling for parameters of neo-vascularization. Moreover, sampling of media following modeled transplantation showed secretory profiles that were correlated with imaging analyses as well as with islet function using glucose-stimulated insulin secretion. Together, evidence revealed that heterotopic constructs consisting of islets, EC, and MSC exhibited the most rapid recruitment and robust branching of cells from the vascular beds suggesting enhanced neo-vascularization compared to islets alone and control constructs. Together, this evidence supports a promising cell transplantation strategy for T1D and also demonstrates a valuable tool for rapidly investigating candidate cellular therapies for transplantation.

    更新日期:2019-11-30
  • Substrate-based kinase activity inference identifies MK2 as driver of colitis
    Integr. Biol. (IF 2.774) Pub Date : 2019-10-16
    Strasser S, Ghazi P, Starchenko A, et al.

    Inflammatory bowel disease (IBD) is a chronic and debilitating disorder that has few treatment options due to a lack of comprehensive understanding of its molecular pathogenesis. We used multiplexed mass spectrometry to collect high-content information on protein phosphorylation in two different mouse models of IBD. Because the biological function of the vast majority of phosphorylation sites remains unknown, we developed Substrate-based Kinase Activity Inference (SKAI), a methodology to infer kinase activity from phosphoproteomic data. This approach draws upon prior knowledge of kinase-substrate interactions to construct custom lists of kinases and their respective substrate sites, termed kinase-substrate sets that employ prior knowledge across organisms. This expansion as much as triples the amount of prior knowledge available. We then used these sets within the Gene Set Enrichment Analysis framework to infer kinase activity based on increased or decreased phosphorylation of its substrates in a dataset. When applied to the phosphoproteomic datasets from the two mouse models, SKAI predicted largely non-overlapping kinase activation profiles. These results suggest that chronic inflammation may arise through activation of largely divergent signaling networks. However, the one kinase inferred to be activated in both mouse models was mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase that functions downstream of p38 stress-activated mitogen-activated protein kinase. Treatment of mice with active colitis with ATI450, an orally bioavailable small molecule inhibitor of the MK2 pathway, reduced inflammatory signaling in the colon and alleviated the clinical and histological features of inflammation. These studies establish MK2 as a therapeutic target in IBD and identify ATI450 as a potential therapy for the disease.

    更新日期:2019-11-27
  • Identification of putative adhesins and carbohydrate ligands of Lactobacillus paracasei using a combinatorial in silico and glycomics microarray profiling approach
    Integr. Biol. (IF 2.774) Pub Date : 2019-11-11
    Houeix B, Synowsky S, Cairns M, et al.

    Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 → 3)-linked sialic acid and α-(1 → 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT.

    更新日期:2019-11-27
  • Adhesive dynamics simulations quantitatively predict effects of kindlin-3 deficiency on T-cell homing
    Integr. Biol. (IF 2.774) Pub Date : 2019-07-22
    Anderson N, Lee D, Hammer D.

    Leukocyte adhesion is important for the proper functioning of the immune system. While leukocyte homing is mediated by adhesion receptors, the activation of these receptors is modulated by intracellular signaling molecules. In Leukocyte Adhesion Deficiency Type 3, the loss of the kindlin-3 prevents the activation of Leukocyte Function-associated Antigen-1 (LFA-1), which leads to a defect in adhesion, causing recurrent infections and bleeding disorders. Here, we use Integrated Signaling Adhesive Dynamics, a computer model of leukocyte rolling and adhesion combined with a simulated intracellular signaling cascade, to predict the response of T cells to depletion of kindlin-3. Our model predicts that cell adhesion is hypersensitive to the amount of kindlin-3 in the cell, while the rolling velocity is independent of kindlin-3 concentration. In addition, our simulation predicted that the time to stop, an important metric of adhesion, would increase with decreasing kindlin-3 expression. These predictions were confirmed experimentally in experiments using Jurkat cells with reduced expression of kindlin-3. These results suggest that Adhesive Dynamics is a versatile tool for quantifying adhesion in the immune response and predicting the effects of engineering cellular components.

    更新日期:2019-08-09
  • Electrical stimulation of cell growth and neurogenesis using conductive and nonconductive microfibrous scaffolds
    Integr. Biol. (IF 2.774) Pub Date : 2019-07-19
    Grossemy S, Chan P, Doran P.

    The effect of exogenous electrical stimulation on cell viability, attachment, growth, and neurogenesis was examined using PC12 cells in microfibrous viscose-rayon scaffolds immersed in culture medium. The scaffolds were applied either in their nonconductive state or after coating the fibres with 200 nm of gold to give a scaffold sheet resistivity of (13 ± 1.3) Ω square−1. The cells were treated for 12 days using direct current electrical stimulation of 2 h per day. No cytotoxic effects were observed when up to 500 mV (8.3 mV mm−1) was applied to the scaffolds without gold, or when up to 100 mV (1.7 mV mm−1) was applied to the scaffolds with gold. Compared with unstimulated cells, whereas electrical stimulation significantly enhanced cell growth and attachment in the nonconductive scaffolds without gold, similar effects were not found for the conductive scaffolds with gold. Neural differentiation in the presence of nerve growth factor was improved by electrical stimulation in both scaffolds; however, neurite development and the expression of key differentiation markers were greater in the nonconductive scaffolds without gold than in the scaffolds with gold. Application of the same current to scaffolds with and without gold led to much higher levels of neurogenesis in the scaffolds without gold. This work demonstrates that substantial benefits in terms of cell growth and neural differentiation can be obtained using electric fields exerted across nonconductive microfibrous scaffolds, and that this approach to electrical stimulation can be more effective than when the stimulus is applied to cells on conductive scaffolds.

    更新日期:2019-08-09
  • Emergent spatiotemporal dynamics of the actomyosin network in the presence of chemical gradients
    Integr. Biol. (IF 2.774) Pub Date : 2019-07-31
    Miller C, LaFosse P, Asokan S, et al.

    We used particle-based computer simulations to study the emergent properties of the actomyosin cytoskeleton. Our model accounted for biophysical interactions between filamentous actin and non-muscle myosin II and was motivated by recent experiments demonstrating that spatial regulation of myosin activity is required for fibroblasts responding to spatial gradients of platelet derived growth factor (PDGF) to undergo chemotaxis. Our simulations revealed the spontaneous formation of actin asters, consistent with the punctate actin structures observed in chemotacting fibroblasts. We performed a systematic analysis of model parameters to identify biochemical steps in myosin activity that significantly affect aster formation and performed simulations in which model parameter values vary spatially to investigate how the model responds to chemical gradients. Interestingly, spatial variations in motor stiffness generated time-dependent behavior of the actomyosin network, in which actin asters continued to spontaneously form and dissociate in different regions of the gradient. Our results should serve as a guide for future experimental investigations.

    更新日期:2019-08-09
  • NRF2 activates a partial epithelial-mesenchymal transition and is maximally present in a hybrid epithelial/mesenchymal phenotype
    Integr. Biol. (IF 2.774) Pub Date : 2019-07-22
    Bocci F, Tripathi S, Vilchez Mercedes S, et al.

    The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype – a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) – the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the ‘metastatic sweet spot’.

    更新日期:2019-08-09
  • Actin stress fiber dynamics in laterally confined cells
    Integr. Biol. (IF 2.774) Pub Date : 2019-07-12
    Müller A, Müller S, Nasufovic V, et al.

    Multiple cellular processes are affected by spatial constraints from the extracellular matrix and neighboring cells. In vitro experiments using defined micro-patterning allow for in-depth analysis and a better understanding of how these constraints impact cellular behavior and functioning. Herein we focused on the analysis of actin cytoskeleton dynamics as a major determinant of mechanotransduction mechanisms in cells. We seeded primary human umbilical vein endothelial cells onto stripe-like cell-adhesive micro-patterns with varying widths and then monitored and quantified the dynamic reorganization of actin stress fibers, including fiber velocities, orientation and density, within these live cells using the cell permeable F-actin marker SiR-actin. Although characteristic parameters describing the overall stress fiber architecture (average orientation and density) were nearly constant throughout the observation time interval of 60 min, we observed permanent transport and turnover of individual actin stress fibers. Stress fibers were more strongly oriented along stripe direction with decreasing stripe width, (5° on 20 μm patterns and 10° on 40 μm patterns), together with an overall narrowing of the distribution of fiber orientation. Fiber dynamics was characterized by a directed movement from the cell edges towards the cell center, where fiber dissolution frequently took place. By kymograph analysis, we found median fiber velocities in the range of 0.2 μm/min with a weak dependence on pattern width. Taken together, these data suggest that cell geometry determines actin fiber orientation, while it also affects actin fiber transport and turnover.

    更新日期:2019-07-25
  • Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-28
    Schultzhaus J, Dean S, Leary D, et al.

    Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

    更新日期:2019-07-25
  • 3D collagen architecture regulates cell adhesion through degradability, thereby controlling metabolic and oxidative stress
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-28
    Velez D, Ranamukhaarachchi S, Kumar A, et al.

    The collagen-rich tumor microenvironment plays a critical role in directing the migration behavior of cancer cells. 3D collagen architectures with small pores have been shown to confine cells and induce aggressive collective migration, irrespective of matrix stiffness and density. However, it remains unclear how cells sense collagen architecture and transduce this information to initiate collective migration. Here, we tune collagen architecture and analyze its effect on four core cell-ECM interactions: cytoskeletal polymerization, adhesion, contractility, and matrix degradation. From this comprehensive analysis, we deduce that matrix architecture initially modulates cancer cell adhesion strength, and that this results from architecture-induced changes to matrix degradability. That is, architectures with smaller pores are less degradable, and degradability is required for cancer cell adhesion to 3D fibrilar collagen. The biochemical consequences of this 3D low-attachment state are similar to those induced by suspension culture, including metabolic and oxidative stress. One distinction from suspension culture is the induction of collagen catabolism that occurs in 3D low-attachment conditions. Cells also upregulate Snail1 and Notch signaling in response to 3D low-attachment, which suggests a mechanism for the emergence of collective behaviors.

    更新日期:2019-07-25
  • Ranking migration cue contributions to guiding individual fibroblasts faced with a directional decision in simple microfluidic bifurcations
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-28
    Pham Q, Tong A, Rodrigues L, et al.

    Directed cell migration in complex micro-environments, such as in vivo pores, is important for predicting locations of artificial tissue growth and optimizing scaffold architectures. Yet, the directional decisions of cells facing multiple physiochemical cues have not been characterized. Hence, we aim to provide a ranking of the relative importance of the following cues to the decision-making of individual fibroblast cells: chemoattractant concentration gradient, channel width, mitosis, and contact-guidance. In this study, bifurcated micro-channels with branches of different widths were created. Fibroblasts were then allowed to travel across these geometries by following a gradient of platelet-derived growth factor-BB (PDGF-BB) established inside the channels. Subsequently, a combination of statistical analysis and image-based diffusion modeling was used to report how the presence of multiple complex migration cues, including cell-cell influences, affect the fibroblast decision-making. It was found that the cells prefer wider channels over a higher chemoattractant gradient when choosing between asymmetric bifurcated branches. Only when the branches were symmetric in width did the gradient become predominant in directing which path the cell will take. Furthermore, when both the gradient and the channels were symmetric, contact guidance became important for guiding the cells in making directional choices. Based on these results we were able to rank these directional cues from most influential to the least as follows: mitosis > channel width asymmetry > chemoattractant gradient difference > and contact-guidance. It is expected that these results will benefit the fields of regenerative medicine, wound healing and developmental biology.

    更新日期:2019-07-25
  • Studying Parkinson’s disease using Caenorhabditis elegans models in microfluidic devices
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-07
    Youssef K, Tandon A, Rezai P.

    Parkinson’s disease (PD) is a progressive neurological disorder associated with the loss of dopaminergic neurons (DNs) in the substantia nigra and the widespread accumulation of α-synuclein (α-syn) protein, leading to motor impairments and eventual cognitive dysfunction. In-vitro cell cultures and in-vivo animal models have provided the opportunity to investigate the PD pathological hallmarks and identify different therapeutic compounds. However, PD pathogenesis and causes are still not well understood, and effective inhibitory drugs for PD are yet to be discovered. Biologically simple but pathologically relevant disease models and advanced screening technologies are needed to reveal the mechanisms underpinning protein aggregation and PD progression. For instance, Caenorhabditis elegans (C. elegans) offers many advantages for fundamental PD neurobehavioral studies including a simple, well-mapped, and accessible neuronal system, genetic homology to humans, body transparency and amenability to genetic manipulation. Several transgenic worm strains that exhibit multiple PD-related phenotypes have been developed to perform neuronal and behavioral assays and drug screening. However, in conventional worm-based assays, the commonly used techniques are equipment-intensive, slow and low in throughput. Over the past two decades, microfluidics technology has contributed significantly to automation and control of C. elegans assays. In this review, we focus on C. elegans PD models and the recent advancements in microfluidic platforms used for manipulation, handling and neurobehavioral screening of these models. Moreover, we highlight the potential of C. elegans to elucidate the in-vivo mechanisms of neuron-to-neuron protein transfer that may underlie spreading Lewy pathology in PD, and its suitability for in-vitro studies. Given the advantages of C. elegans and microfluidics technology, their integration has the potential to facilitate the investigation of disease pathology and discovery of potential chemical leads for PD.

    更新日期:2019-07-25
  • Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-28
    Williams E, García J, Mannino R, et al.

    Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.

    更新日期:2019-07-05
  • Microfluidic platform for studying osteocyte mechanoregulation of breast cancer bone metastasis
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-24
    Mei X, Middleton K, Shim D, et al.

    Bone metastasis is a common, yet serious, complication of breast cancer. Breast cancer cells that extravasate from blood vessels to the bone devastate bone quality by interacting with bone cells and disrupting the bone remodeling balance. Although exercise is often suggested as a cancer intervention strategy and mechanical loading during exercise is known to regulate bone remodeling, its role in preventing bone metastasis remains unknown. We developed a novel in vitro microfluidic tissue model to investigate the role of osteocytes in the mechanical regulation of breast cancer bone metastasis. Metastatic MDA-MB-231 breast cancer cells were cultured inside a 3D microfluidic lumen lined with human umbilical vein endothelial cells (HUVECs), which is adjacent to a channel seeded with osteocyte-like MLO-Y4 cells. Physiologically relevant oscillatory fluid flow (OFF) (1 Pa, 1 Hz) was applied to mechanically stimulate the osteocytes. Hydrogel-filled side channels in-between the two channels allowed real-time, bi-directional cellular signaling and cancer cell extravasation over 3 days. The applied OFF was capable of inducing intracellular calcium responses in osteocytes (82.3% cells responding with a 3.71 fold increase average magnitude). Both extravasation distance and percentage of extravasated side-channels were significantly reduced with mechanically stimulated osteocytes (32.4% and 53.5% of control, respectively) compared to static osteocytes (102.1% and 107.3% of control, respectively). This is the first microfluidic device that has successfully integrated stimulatory bone fluid flow, and demonstrated that mechanically stimulated osteocytes reduced breast cancer extravasation. Future work with this platform will determine the specific mechanisms involved in osteocyte mechanoregulation of breast cancer bone metastasis, as well as other types of cancer metastasis and diseases.

    更新日期:2019-07-05
  • Local cell coordination does not alter individual cell migration during collective migration but does impact cellular exchange events
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-31
    Slater B, Ng E, McGuigan A.

    Coordinated cell re-organization is critical to ensure correct tissue morphogenesis for a number of important embryonic and tissue repair events, however the mechanisms that govern cells coordination during collective movements, particularly in situations where cells are spatially restricted by their neighbours, are not well understood. Here we assessed cell re-organization in monolayers of retinal epithelial cells (ARPE-19) to determine if cells that coordinate with their neighbours exhibit differential migration properties to non-coordinating cells and participate differently in local cell re-organization of the tissue sheet. From global tracking analysis, we determined that the movement profiles of cells were indistinguishable regardless of whether or not they were a part of multicellular streams. Using high magnification live imaging of cell membranes, we also characterized the localized geometry and organization of a monolayer (cell area, number of nearest neighbours, aspect ratio, internal cell angles) during cell re-organization in both streaming and non-streaming regions. Consistent with our global migration analysis, we observed no differences in cell sheet geometry and organization in streaming versus non-streaming regions. We did however observe that cells executed T1-like transitions to exchange position within the space-limited monolayer and that exchange events consistently involved at least one non-streaming cell. Our data suggests a model in which cell movement within the sheet is limited by neighbour exchange events and likely cells transition between streaming and non-streaming regimes to facilitate these neighbour exchange events while maintaining the integrity of the sheet.

    更新日期:2019-07-05
  • Long-term fluorescence hyperspectral imaging of on-chip treated co-culture tumour spheroids to follow clonal evolution
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-07
    St-Georges-Robillard A, Cahuzac M, Péant B, et al.

    Multicellular tumour spheroids are an ideal in vitro tumour model to study clonal heterogeneity and drug resistance in cancer research because different cell types can be mixed at will. However, measuring the individual response of each cell population over time is challenging: current methods are either destructive, such as flow cytometry, or cannot image throughout a spheroid, such as confocal microscopy. Our group previously developed a wide-field fluorescence hyperspectral imaging system to study spheroids formed and cultured in microfluidic chips. In the present study, two subclones of a single parental ovarian cancer cell line transfected to express different fluorophores were produced and co-culture spheroids were formed on-chip using ratios forming highly asymmetric subpopulations. We performed a 3D proliferation assay on each cell population forming the spheroids that matched the 2D growth behaviour. Response assays to PARP inhibitors and platinum-based drugs were also performed to follow the clonal evolution of mixed populations. Our experiments show that hyperspectral imaging can detect spheroid response before observing a decrease in spheroid diameter. Hyperspectral imaging and microfluidic-based spheroid assays provide a versatile solution to study clonal heterogeneity, able to measure response in subpopulations presenting as little as 10% of the initial spheroid.

    更新日期:2019-07-05
  • Microfluidic platform enables live-cell imaging of signaling and transcription combined with multiplexed secretion measurements in the same single cells
    Integr. Biol. (IF 2.774) Pub Date : 2019-06-26
    Ramji R, Alexander A, Muñoz-Rojas A, et al.

    Innate immune cells, including macrophages and dendritic cells, protect the host from pathogenic assaults in part through secretion of a program of cytokines and chemokines (C/Cs). Cell-to-cell variability in C/C secretion appears to contribute to the regulation of the immune response, but the sources of secretion variability are largely unknown. To begin to track the biological sources that control secretion variability, we developed and validated a microfluidic device to integrate live-cell imaging of fluorescent reporter proteins with a single-cell assay of protein secretion. We used this device to image NF-κB RelA nuclear translocation dynamics and Tnf transcription dynamics in macrophages in response to stimulation with the bacterial component lipopolysaccharide (LPS), followed by quantification of secretion of TNF, CCL2, CCL3, and CCL5. We found that the timing of the initial peak of RelA signaling in part determined the relative level of TNF and CCL3 secretion, but not CCL2 and CCL5 secretion. Our results support evidence that differences in timing across cell processes partly account for cell-to-cell variability in downstream responses, but that other factors introduce variability at each biological step.

    更新日期:2019-07-05
  • Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection
    Integr. Biol. (IF 2.774) Pub Date : 2019-03-30
    Pulkkinen E, Hinkley T, Nugen S.

    Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

    更新日期:2019-05-21
  • Protein dynamic analysis of the budding yeast sporulation process at the single-cell level in an air-enriched microfluidic device
    Integr. Biol. (IF 2.774) Pub Date : 2019-04-10
    Zhao X, Luo C, Wang H.

    The sporulation process of the budding yeast Saccharomyces cerevisiae involves morphological changes, which is a complex and interesting process in yeast biology. However, efficient methods to accurately study protein dynamic behaviors in the sporulation process are still lacking because the cell behaviors are quite heterogeneous. In this study, we proposed the use of an air-enriched microfluidic system to investigate protein dynamics during sporulation of the budding yeast S. cerevisiae within single cells. Our results verified that the air-enriched microenvironment was important for the yeast sporulation process in the microfluidic system. Furthermore, we found that the sporulation temporal stage of different cells varied immensely, indicating that single-cell analysis instead of statistical analysis of a population was necessary to verify the accuracy of protein dynamics. In addition, we developed a ‘data synchronization’ approach. This approach aligned each cell on a time line according to its own morphological changes. Using these methods, we obtained the dynamic performance of six proteins involved in the yeast sporulation process with single cells.

    更新日期:2019-05-21
  • Microdroplet co-cultivation and interaction characterization of human vaginal bacteria
    Integr. Biol. (IF 2.774) Pub Date : 2019-04-11
    Jackman C, Deans K, Forney L, et al.

    The human vaginal microbiome (HVM) plays a fundamental role in women’s reproductive health. For instance, bacterial vaginosis (BV) is characterized by a depletion of lactobacilli and an overgrowth of strict anaerobes. Women with BV may have an increased risk of acquiring sexually transmitted diseases and adverse pregnancy outcomes. Although the HVM is important, the ecological roles of many vaginal species remain unclear and current approaches for investigating them have severe limitations. We previously developed a new high-throughput technology based on the co-cultivation of bacteria in microdroplets to dissect inter-species interactions in microbial communities. Here, we adapted and extended this technology to investigate the HVM and tested it using pairwise model systems. In one case, Lactobacillus jensenii JV-V16, a lactic acid bacterium, and Gardnerella vaginalis ATCC 49145, a bacterium associated with BV, were cultured in microdroplets as pure cultures and co-cultures. Two assays were developed to analyze their growth in microdroplets. First, qPCR was used to quantify the bacteria in pooled microdroplets. Second, cells in individual microdroplets were plated and enumerated on agar media. The results showed that growth of G. vaginalis was severely inhibited by L. jensenii, which recapitulated previous findings of studies conducted in flask batch cultures. Additionally, we validated the general applicability of our technology pipeline with a second co-culture model system by observing that Enterococcus faecalis, another bacterium from the urogenital tract, was also inhibited by L. jensenii. Our results show that co-cultivation and characterization of bacteria in microdroplets provides an effective way to study inter-species interactions in microbial ecosystems.

    更新日期:2019-05-21
  • Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-13
    Stolwijk J, Skiba M, Kade C, et al.

    Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample’s impedance, we were able to establish a full concentration–response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

    更新日期:2019-05-21
  • Glycation of collagen matrices promotes breast tumor cell invasion
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-01
    Suh Y, Hall M, Huang Y, et al.

    Cancer metastasis is a physical process in which tumor cells break away from the primary tumor, enter, and then exit the blood or lymph vessels, and establish secondary tumors in distant organs. Current clinical studies report a higher risk of cancer metastasis for diabetics than non-diabetics. However, due to complex overlapping risk factors between diabetes and cancer, the mechanism underlying this correlation is largely unknown. Elevated lifetime blood sugar levels in diabetics are known to increase glycation of collagen, causing stiffening of the ECM and connective tissue. In this study, we explored the roles of glycation of 3D collagen matrices in tumor cell invasion and migration. Using time-lapse images, we quantitatively compared the motility behavior of malignant breast tumor cells (MDA-MB-231) and co-culture spheroids (1:1 ratio of MDA-MB-231 cells with normal epithelial MCF-10A cells) embedded in glycated and non-glycated collagen matrices of various concentrations. Experimental results demonstrated that glycation increased tumor invasion within collagen matrices. More specifically, the average speed of MDA-MB-231 cells was higher in glycated collagen gels than in non-glycated collagen gels for all three gel concentrations tested. Cell spreading characterized by its diffusion coefficient or the effective spheroid radii at various time points was significantly greater in glycated collagen than in non-glycated collagen at a concentration of 3.5 mg/mL. This enhancement was moderate and less evident at lower collagen concentrations of 1.0 and 2.0 mg/mL. These results suggest a possible biomechanical link that relates to the high blood sugar level in diabetic patients and the cancer metastatic outcome.

    更新日期:2019-05-21
  • Raman spectroscopy reveals LPS-induced changes of biomolecular composition in monocytic THP-1 cells in a label-free manner
    Integr. Biol. (IF 2.774) Pub Date : 2019-05-13
    Töpfer N, Müller M, Dahms M, et al.

    The human innate immune system is able to recognize pathogen-associated molecular patterns like lipopolysaccharides (LPS) leading to the activation of signal cascades and the release of different cytokines. Activation of the immune cells can be assessed in different ways which are either indirect (ELISA of cytokine release), require staining protocols (flow cytometry) or lysis of the cells (mRNA analysis). Here, Raman spectroscopy as a non-destructive spectroscopic method is presented to enable direct and label-free monitoring of changes in cellular metabolism, biomolecular composition as well as morphology. Exemplarily, the potential of Raman spectroscopy is presented for the characterization of LPS-stimulation of monocytic THP-1 cells over a time course of 16 h. The cell culture stimulation model is characterized using gene transcription and expression of the two cytokines TNFα and IL-1β. After 1 h, 3 h, 8 h and 16 h specific Raman spectroscopic fingerprints are generated which encode cell activation pattern after TLR4 stimulation. Most prevalent changes in the spectra occur after 8 h, but slight differences are already detectable after 1 h. Spatially highly resolved Raman scans are used to generate false-color Raman images which provide spatial information of the biochemical state of the cells and changes over time. One of the most significant observed differences is an increase in Raman signal from DNA/RNA content in LPS-stimulated cells when compared to unstimulated cells. The systematic assignment of Raman spectroscopic profiles of LPS-activated cells to cellular activation assessed by cytokine gene transcription and expression opens new ways for label-free and direct immunological studies of specific pathogen recognizing receptors and their downstream signaling pathways.

    更新日期:2019-05-21
  • Rapid translocation of pluripotency-related transcription factors by external uniaxial forces
    Integr. Biol. (IF 2.774) Pub Date : 2019-02-26
    Topal T, Kim B, Villa-Diaz L, et al.

    Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.

    更新日期:2019-05-16
  • Protein–ligand interaction fingerprints for accurate prediction of dissociation rates of p38 MAPK Type II inhibitors
    Integr. Biol. (IF 2.774) Pub Date : 2019-03-11
    Zhang D, Huang S, Mei H, et al.

    Binding/unbinding kinetics are key determinants of drug potencies. However, there are still a lot of challenges in predicting kinetic properties during early-stage drug development. In this work, position-restrained molecular dynamics simulations combined with energy decomposition were applied to extract protein–ligand interaction (PLI) fingerprints along the unbinding pathway of 20 p38 mitogen-activated protein kinase (p38 MAPK) Type II inhibitors. The results showed that the electrostatic and/or van der Waals interaction fingerprints at three key positions can be used for accurate prediction of the dissociation rate constants (koff) of p38 MAPK Type II inhibitors. The strategy proposed in this paper can provide not only an efficient method of predicting the dissociation rates of the p38 MAPK Type II inhibitors, but also the atom-level mechanism of enthalpy-driven unbinding process.

    更新日期:2019-05-16
  • Shear stress induces expression, intracellular reorganization and enhanced Notch activation potential of Jagged1
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-17
    R. C. H. Driessen, O. M. J. A. Stassen, M. Sjöqvist, F. Suarez Rodriguez, J. Grolleman, C. V. C. Bouten, C. M. Sahlgren
    更新日期:2019-02-26
  • Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-15
    Juan Wang, Joseph Koelbl, Anuraag Boddupalli, Zhiqi Yao, Kaitlin M. Bratlie, Ian C. Schneider
    更新日期:2019-02-26
  • Chemical modification of enveloped viruses for biomedical applications
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-08
    Pahweenvaj Ratnatilaka Na Bhuket, Jittima Amie Luckanagul, Pornchai Rojsitthisak, Qian Wang
    更新日期:2019-02-26
  • Engineered basement membranes: from in vivo considerations to cell-based assays
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-08
    Guillaume Perry, Wenjin Xiao, Gavin I. Welsh, Adam W. Perriman, Rachel Lennon
    更新日期:2019-02-26
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  • Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging
    Integr. Biol. (IF 2.774) Pub Date : 2018-09-05
    Catarina Costa Moura, Stuart A. Lanham, Tual Monfort, Konstantinos N. Bourdakos, Rahul S. Tare, Richard O. C. Oreffo, Sumeet Mahajan
    更新日期:2019-02-26
  • Computational model of wound healing: EGF secreted by fibroblasts promotes delayed re-epithelialization of epithelial keratinocytes
    Integr. Biol. (IF 2.774) Pub Date : 2018-09-04
    Vivi Andasari, Dongyuan Lü, Maciej Swat, Shiliang Feng, Fabian Spill, Li Chen, Xiangdong Luo, Muhammad Zaman, Mian Long
    更新日期:2019-02-26
  • Effect of macromolecular crowding on the kinetics of glycolytic enzymes and the behaviour of glycolysis in yeast
    Integr. Biol. (IF 2.774) Pub Date : 2018-09-03
    Henrik S. Thoke, Luis A. Bagatolli, Lars F. Olsen
    更新日期:2019-02-26
  • Direct immunoassays and their performance – theoretical modelling of the effects of antibody orientation and associated kinetics
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-30
    Dana Mackey, Eilís Kelly, Robert Nooney, Richard O'Kennedy
    更新日期:2019-02-26
  • A robust control system for targeting melanoma by a supermolecular DDMC/paclitaxel complex
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-24
    Y. Onishi, Y. Eshita, R.-C. Ji, T. Kobayashi, M. Onishi, M. Mizuno, J. Yoshida, N. Kubota
    更新日期:2019-02-26
  • A high-throughput microtissue platform to probe endothelial function in vitro
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-17
    Alexandra L. Crampton, Katherine A. Cummins, David K. Wood
    更新日期:2019-02-26
  • Adaptive reorientation of endothelial collectives in response to strain
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-06
    Laura Bernardi, Costanza Giampietro, Vita Marina, Martina Genta, Edoardo Mazza, Aldo Ferrari
    更新日期:2019-02-26
  • Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-06
    Julian Michael Rocha, Charles Joseph Richardson, Mingxing Zhang, Caroline Maureen Darch, Eugene Cai, Andreas Diepold, Andreas Gahlmann
    更新日期:2019-02-26
  • Automated screening of C. elegans neurodegeneration mutants enabled by microfluidics and image analysis algorithms
    Integr. Biol. (IF 2.774) Pub Date : 2018-08-01
    Ivan de Carlos Cáceres, Daniel A. Porto, Ivan Gallotta, Pamela Santonicola, Josue Rodríguez-Cordero, Elia Di Schiavi, Hang Lu
    更新日期:2019-02-26
  • Teratogen screening with human pluripotent stem cells
    Integr. Biol. (IF 2.774) Pub Date : 2018-07-31
    Kathryn E. Worley, Jennifer Rico-Varela, Dominic Ho, Leo Q. Wan
    更新日期:2019-02-26
  • Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter
    Integr. Biol. (IF 2.774) Pub Date : 2018-07-30
    Premashis Manna, Sheng-Ting Hung, Srijit Mukherjee, Pia Friis, David M. Simpson, Maria N. Lo, Amy E. Palmer, Ralph Jimenez
    更新日期:2019-02-26
  • Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex
    Integr. Biol. (IF 2.774) Pub Date : 2018-12-06
    Shuo Han, Shiruo Wei, Xuan Wang, Xu Han, Mingzhi Zhang, Ming Su, Yang Li, Jinhai Guo, Wotan Zeng, Jinwen Liu, Yi Gao, Li Shen
    更新日期:2018-12-19
  • Protein structure networks provide insight into active site flexibility in esterase/lipases from the carnivorous plant Drosera capensis
    Integr. Biol. (IF 2.774) Pub Date : 2018-12-05
    Vy T. Duong, Megha H. Unhelkar, John E. Kelly, Suhn H. Kim, Carter T. Butts, Rachel W. Martin
    更新日期:2018-12-19
  • An adhesion based approach for the detection of esophageal cancer
    Integr. Biol. (IF 2.774) Pub Date : 2018-11-06
    Mahboubeh S. Noori, Evan S. Streator, Grady E. Carlson, David S. Drozek, Monica M. Burdick, Douglas J. Goetz
    更新日期:2018-12-19
  • Morphology-based prediction of cancer cell migration using an artificial neural network and a random decision forest
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-29
    Zhixiong Zhang, Lili Chen, Brock Humphries, Riley Brien, Max S. Wicha, Kathryn E. Luker, Gary D. Luker, Yu-Chih Chen, Euisik Yoon
    更新日期:2018-12-19
  • Substrate stiffness heterogeneities disrupt endothelial barrier integrity in a micropillar model of heterogeneous vascular stiffening
    Integr. Biol. (IF 2.774) Pub Date : 2018-10-24
    Jacob A. VanderBurgh, Halie Hotchkiss, Archit Potharazu, Paul V. Taufalele, Cynthia A. Reinhart-King
    更新日期:2018-12-19
  • Real-time detection of stimulus response in cultured neurons by high-intensity intermediate-frequency magnetic field exposure
    Integr. Biol. (IF 2.774) Pub Date : 2018-07-17
    Atsushi Saito, Tatsuya Terai, Kei Makino, Masayuki Takahashi, Sachiko Yoshie, Masateru Ikehata, Yasuhiko Jimbo, Keiji Wada, Yukihisa Suzuki, Satoshi Nakasono
    更新日期:2018-08-13
  • Engineering of a genetic circuit with regulatable multistability
    Integr. Biol. (IF 2.774) Pub Date : 2018-07-05
    Tingting Li, Yiming Dong, Xuanqi Zhang, Xiangyu Ji, Chunxiong Luo, Chunbo Lou, Haoqian M. Zhang, Qi Ouyang
    更新日期:2018-08-13
  • The tactile receptive fields of freely moving Caenorhabditis elegans nematodes
    Integr. Biol. (IF 2.774) Pub Date : 2018-06-27
    E. A. Mazzochette, A. L. Nekimken, F. Loizeau, J. Whitworth, B. Huynh, M. B. Goodman, B. L. Pruitt
    更新日期:2018-08-13
  • Molecular vibration as a novel explanatory mechanism for the expression of animal colouration
    Integr. Biol. (IF 2.774) Pub Date : 2018-06-20
    Ismael Galván, Javier Cerezo, Alberto Jorge, Kazumasa Wakamatsu
    更新日期:2018-08-13
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