Microstructure-based modeling of primary cilia mechanics.

A primary cilium, made of nine microtubule doublets enclosed in a cilium membrane, is a mechanosensing organelle that bends under an external mechanical load and sends an intracellular signal through transmembrane proteins activated by cilium bending. The nine microtubule doublets are the main load-bearing structural component, while the transmembrane proteins on the cilium membrane are the main sensing component. No distinction was made between these two components in all existing models, where the stress calculated from the structural component (nine microtubule doublets) was used to explain the sensing location, which may be totally misleading. For the first time, we developed a microstructure-based primary cilium model by considering these two components separately. First, we refined the analytical solution of bending an orthotropic cylindrical shell for individual microtubule, and obtained excellent agreement between finite element simulations and the theoretical predictions of a microtubule bending as a validation of the structural component in the model. Second, by integrating the cilium membrane with nine microtubule doublets and simulating the tip-anchored optical tweezer experiment on our computational model, we found that the microtubule doublets may twist significantly as the whole cilium bends. Third, besides being cilium-length-dependent, we found the mechanical properties of the cilium are also highly deformation-dependent. More important, we found that the cilium membrane near the base is not under pure in-plane tension or compression as previously thought, but has significant local bending stress. This challenges the traditional model of cilium mechanosensing, indicating that transmembrane proteins may be activated more by membrane curvature than membrane stretching. Finally, we incorporated imaging data of primary cilia into our microstructure-based cilium model, and found that comparing to the ideal model with uniform microtubule length, the imaging-informed model shows the nine microtubule doublets interact more evenly with the cilium membrane, and their contact locations can cause even higher bending curvature in the cilium membrane than near the base.

Microstructure-based nuclear lamina constitutive model.

The nuclear lamina is widely recognized as the most crucial component in providing mechanical stability to the nucleus. However, it is still a significant challenge to model the mechanics of this multilayered protein network. We developed a constitutive model of the nuclear lamina network based on its microstructure, which accounts for the deformation phases at the dimer level, as well as the orientational arrangement and density of lamin filaments. Instead of relying on homology modeling in the previous studies, we conducted molecular simulations to predict the force-extension response of a highly accurate lamin dimer structure obtained through X-ray diffraction crystallography experimentation. Furthermore, we devised a semiflexible worm-like chain extension-force model of lamin dimer as a substitute, incorporating phases of initial stretching, uncoiling of the dimer coiled-coil, and transition of secondary structures. Subsequently, we developed a 2D network continuum model for the nuclear lamina by using our extension-force lamin dimer model and derived stress resultants. By comparing with experimentally measured lamina modulus, we found that the lamina network has sharp initial strain-hardening behavior. This also enabled us to carry out finite element simulations of the entire nucleus with an accurate microstructure-based nuclear lamina model. Finally, we conducted simulations of transendothelial transmigration of a nucleus and investigated the impact of varying network density and uncoiling constants on the critical force required for successful transmigration. The model allows us to incorporate the microstructure characteristics of the nuclear lamina into the nucleus model, thereby gaining insights into how laminopathies and mutations affect nuclear mechanics.

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Increased expression of Mycobacterium tuberculosis Rv3737 gene associated with low-level amikacin resistance.

INTRODUCTION: As an infectious disease, tuberculosis (TB) poses a serious threat to public health. Although amikacin (AMK) is an important antibiotic for the treatment of drug-resistant TB, its resistance mechanisms are not fully understood. METHODS: To investigate the role of Rv3737 gene on AMK drug susceptibility, a Mycobacterium tuberculosis (M.tb) Rv3737 knockout strain (H37Rv△Rv3737) and a Mycobacterium smegmatis (M.sm) Rv3737 overexpressing strain (Msm/pMV261-Rv3737) were used to detect their minimal inhibitory concentrations (MICs) in this study. RESULTS: The AMK MICs of Rv3737 knockout and overexpressing strains were 4-fold lower and 2-fold higher than those of the wild-type and empty plasmid strains, respectively. The results of clinical isolates showed that no Rv3737 gene mutation was found to be associated with AMK susceptibility, while the rrs A1401G mutation remained the main mechanism of high level of AMK resistance (MIC>32 µg/ml). There was a positive correlation between Rv3737 mRNA expression level and AMK MIC. In the isolates with low-level AMK resistance (MIC = 4 µg/ml) without rrs A1401G mutation, the expression level of Rv3737 gene was significantly higher than those of susceptible isolates. CONCLUSIONS: In this study, the Rv3737 gene was reported for the first time for its effect on AMK susceptibility in M.tb. Although the rrs A1401G mutation remains the main reason of high-level AMK resistance, high expression of the Rv3737 gene was associated with low-level AMK resistance in clinical isolates.

Polyurethane Culture Substrates Enable Long-Term Neuron Monoculture in a Human in vitro Model of Neurotrauma.

Human induced pluripotent stem cell (hiPSC)-derived cells can reproduce human-specific pathophysiology, patient-specific vulnerability, and gene-environment interactions in neurological disease. Human in vitro models of neurotrauma therefore have great potential to advance the field. However, this potential cannot be realized until important biomaterials challenges are addressed. Status quo stretch injury models of neurotrauma culture cells on sheets of polydimethylsiloxane (PDMS) that are incompatible with long-term monoculture of hiPSC-derived neurons. Here, we overcame this challenge in an established human in vitro neurotrauma model by replacing PDMS with a highly biocompatible form of polyurethane (PU). This substitution allowed long-term monoculture of hiPSC-derived neurons. It also changed the biomechanics of stretch injury. We quantified these changes experimentally using high-speed videography and digital image correlation. We used finite element modeling to quantify the influence of the culture substrate's thickness, stiffness, and coefficient of friction on membrane stretch and concluded that the coefficient of friction explained most of the observed biomechanical changes. Despite these changes, we demonstrated that the modified model produced a robust, dose-dependent trauma phenotype in hiPSC-derived neuron monocultures. In summary, the introduction of this PU film makes it possible to maintain hiPSC-derived neurons in monoculture for long periods in a human in vitro neurotrauma model. In doing so, it opens new horizons in the field of neurotrauma by enabling the unique experimental paradigms (e.g., isogenic models) associated with hiPSC-derived neurons.

Transit Time Theory for a Droplet Passing through a Slit in Pressure-Driven Low Reynolds Number Flows.

Soft objects squeezing through small apertures are crucial for many in vivo and in vitro processes. Red blood cell transit time through splenic inter-endothelial slits (IESs) plays a crucial role in blood filtration and disease progression, while droplet velocity through constrictions in microfluidic devices is important for effective manipulation and separation processes. As these transit phenomena are not well understood, we sought to establish analytical and numerical solutions of viscous droplet transit through a rectangular slit. This study extends from our former theory of a circular pore because a rectangular slit is more realistic in many physiological and engineering applications. Here, we derived the ordinary differential equations (ODEs) of a droplet passing through a slit by combining planar Poiseuille flow, the Young-Laplace equations, and modifying them to consider the lubrication layer between the droplet and the slit wall. Compared to the pore case, we used the Roscoe solution instead of the Sampson one to account for the flow entering and exiting a rectangular slit. When the surface tension and lubrication layer were negligible, we derived the closed-form solutions of transit time. When the surface tension and lubrication layer were finite, the ODEs were solved numerically to study the impact of various parameters on the transit time. With our solutions, we identified the impact of prescribed pressure drop, slit dimensions, and droplet parameters such as surface tension, viscosity, and volume on transit time. In addition, we also considered the effect of pressure drop and surface tension near critical values. For this study, critical surface tension for a given pressure drop describes the threshold droplet surface tension that prevents transit, and critical pressure for a given surface tension describes the threshold pressure drop that prevents transit. Our solutions demonstrate that there is a linear relationship between pressure and the reciprocal of the transit time (referred to as inverse transit time), as well as a linear relationship between viscosity and transit time. Additionally, when the droplet size increases with respect to the slit dimensions, there is a corresponding increase in transit time. Most notably, we emphasize the initial antagonistic effect of surface tension which resists droplet passage but at the same time decreases the lubrication layer, thus facilitating passage. Our results provide quantitative calculations for understanding cells passing through slit-like constrictions and designing droplet microfluidic experiments.

Physical mechanisms of red blood cell splenic filtration.

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 [Formula: see text]m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Numerical Modeling of Physical Cell Trapping in Microfluidic Chips.

Microfluidic methods have proven to be effective in separation and isolation of cells for a wide range of biomedical applications. Among these methods, physical trapping is a label-free isolation approach that relies on cell size as the selective phenotype to retain target cells on-chip for follow-up analysis and imaging. In silico models have been used to optimize the design of such hydrodynamic traps and to investigate cancer cell transmigration through narrow constrictions. While most studies focus on computational fluid dynamics (CFD) analysis of flow over cells and/or pillar traps, a quantitative analysis of mechanical interaction between cells and trapping units is missing. The existing literature centers on longitudinally extended geometries (e.g., micro-vessels) to understand the biological phenomenon rather than designing an effective cell trap. In this work, we aim to make an experimentally informed prediction of the critical pressure for a cell to pass through a trapping unit as a function of cell morphology and trapping unit geometry. Our findings show that a hyperelastic material model accurately captures the stress-related softening behavior observed in cancer cells passing through micro-constrictions. These findings are used to develop a model capable of predicting and extrapolating critical pressure values. The validity of the model is assessed with experimental data. Regression analysis is used to derive a mathematical framework for critical pressure. Coupled with CFD analysis, one can use this formulation to design efficient microfluidic devices for cell trapping and potentially perform downstream analysis of trapped cells.

Microstructure-Based Modeling of Primary Cilia Mechanics.

A primary cilium, made of nine microtubule doublets enclosed in a cilium membrane, is a mechanosensing organelle that bends under an external mechanical load and sends an intracellular signal through transmembrane proteins activated by cilium bending. The nine microtubule doublets are the main load-bearing structural component, while the transmembrane proteins on the cilium membrane are the main sensing component. No distinction was made between these two components in all existing models, where the stress calculated from the structural component (nine microtubule doublets) was used to explain the sensing location, which may be totally misleading. For the first time, we developed a microstructure-based primary cilium model by considering these two components separately. First, we refined the analytical solution of bending an orthotropic cylindrical shell for individual microtubule, and obtained excellent agreement between finite element simulations and the theoretical predictions of a microtubule bending as a validation of the structural component in the model. Second, by integrating the cilium membrane with nine microtubule doublets, we found that the microtubule doublets may twist significantly as the whole cilium bends. Third, besides being cilium-length-dependent, we found the mechanical properties of the cilium are also highly deformation-dependent. More important, we found that the cilium membrane near the base is not under pure in-plane tension or compression as previously thought, but has significant local bending stress. This challenges the traditional model of cilium mechanosensing, indicating that transmembrane proteins may be activated more by membrane curvature than membrane stretching. Finally, we incorporated imaging data of primary cilia into our microstructure-based cilium model, and found that comparing to the ideal model with uniform microtubule length, the imaging-informed model shows the nine microtubule doublets interact more evenly with the cilium membrane, and their contact locations can cause even higher bending curvature in the cilium membrane than near the base. SIGNIFICANCE: Factors regulating the mechanical response of a primary cilium to fluid flow remain unclear. Modeling the microtubule doublet as a composite of two orthotropic shells and the ciliary axoneme as an elastic shell enclosing nine such microtubule doublets, we found that the length distribution of microtubule doublets (inferred from cryogenic electron tomography images) is the primary determining factor in the bending stiffness of primary cilia, rather than just the ciliary length. This implies ciliary-associated transmembrane proteins may be activated by membrane curvature changes rather than just membrane stretching. These insights challenge the traditional view of ciliary mechanosensation and expands our understanding of the different ways in which cells perceive and respond to mechanical stimuli.

Evolution of focused streams for viscoelastic flow in spiral microchannels.

Particle migration dynamics in viscoelastic fluids in spiral channels have attracted interest in recent years due to potential applications in the 3D focusing and label-free sorting of particles and cells. Despite a number of recent studies, the underlying mechanism of Dean-coupled elasto-inertial migration in spiral microchannels is not fully understood. In this work, for the first time, we experimentally demonstrate the evolution of particle focusing behavior along a channel downstream length at a high blockage ratio. We found that flow rate, device curvature, and medium viscosity play important roles in particle lateral migration. Our results illustrate the full focusing pattern along the downstream channel length, with side-view imaging yielding observations on the vertical migration of focused streams. Ultimately, we anticipate that these results will offer a useful guide for elasto-inertial microfluidics device design to improve the efficiency of 3D focusing in cell sorting and cytometry applications.

Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity.

Various signals in tissue microenvironments are often unevenly distributed around cells. Cellular responses to asymmetric cell-matrix adhesion in a 3D space remain generally unclear and are to be studied at the single-cell resolution. Here, the authors developed a droplet-based microfluidic approach to manufacture a pure population of single cells in a microscale layer of compartmentalized 3D hydrogel matrices with a tunable spatial presentation of ligands at the subcellular level. Cells elongate with an asymmetric presentation of the integrin adhesion ligand Arg-Gly-Asp (RGD), while cells expand isotropically with a symmetric presentation of RGD. Membrane tension is higher on the side of single cells interacting with RGD than on the side without RGD. Finite element analysis shows that a non-uniform isotropic cell volume expansion model is sufficient to recapitulate the experimental results. At a longer timescale, asymmetric ligand presentation commits mesenchymal stem cells to the osteogenic lineage. Cdc42 is an essential mediator of cell polarization and lineage specification in response to asymmetric cell-matrix adhesion. This study highlights the utility of precisely controlling 3D ligand presentation around single cells to direct cell polarity for regenerative engineering and medicine.

Elasto-Inertial Focusing Mechanisms of Particles in Shear-Thinning Viscoelastic Fluid in Rectangular Microchannels.

Growth of the microfluidics field has triggered numerous advances in focusing and separating microparticles, with such systems rapidly finding applications in biomedical, chemical, and environmental fields. The use of shear-thinning viscoelastic fluids in microfluidic channels is leading to evolution of elasto-inertial focusing. Herein, we showed that the interplay between the elastic and shear-gradient lift forces, as well as the secondary flow transversal drag force that is caused by the non-zero second normal stress difference, lead to different particle focusing patterns in the elasto-inertial regime. Experiments and 3D simulations were performed to study the effects of flowrate, particle size, and the shear-thinning extent of the fluid on the focusing patterns. The Giesekus constitutive equation was used in the simulations to capture the shear-thinning and viscoelastic behaviors of the solution used in the experiments. At low flowrate, with Weissenberg number Wi ~ O(1), both the elastic force and secondary flow effects push particles towards the channel center. However, at a high flowrate, Wi ~ O(10), the elastic force direction is reversed in the central regions. This remarkable behavior of the elastic force, combined with the enhanced shear-gradient lift at the high flowrate, pushes particles away from the channel center. Additionally, a precise prediction of the focusing position can only be made when the shear-thinning extent of the fluid is correctly estimated in the modeling. The shear-thinning also gives rise to the unique behavior of the inertial forces near the channel walls which is linked with the 'warped' velocity profile in such fluids.

Cyclic Mechanical Stresses Alter Erythrocyte Membrane Composition and Microstructure and Trigger Macrophage Phagocytosis.

Red blood cells (RBCs) are cleared from the circulation when they become damaged or display aging signals targeted by macrophages. This process occurs mainly in the spleen, where blood flows through submicrometric constrictions called inter-endothelial slits (IES), subjecting RBCs to large-amplitude deformations. In this work, RBCs are circulated through microfluidic devices containing microchannels that replicate the IES. The cyclic mechanical stresses experienced by the cells affect their biophysical properties and molecular composition, accelerating cell aging. Specifically, RBCs quickly transition to a more spherical, less deformable phenotype that hinders microchannel passage, causing hemolysis. This transition is associated with the release of membrane vesicles, which self-extinguishes as the spacing between membrane-cytoskeleton linkers becomes tighter. Proteomics analysis of the mechanically aged RBCs reveals significant losses of essential proteins involved in antioxidant protection, gas transport, and cell metabolism. Finally, it is shown that these changes make mechanically aged RBCs more susceptible to macrophage phagocytosis. These results provide a comprehensive model explaining how physical stress induces RBC clearance in the spleen. The data also suggest new biomarkers of early "hemodamage" and inflammation preceding hemolysis in RBCs subjected to mechanical stress.

Mycobacterial PPE36 Modulates Host Inflammation by Promoting E3 Ligase Smurf1-Mediated MyD88 Degradation.

Mycobacterium tuberculosis (Mtb) PPE36, a cell-wall-associated protein, is highly specific and conserved for the Mtb complex group. Although PPE36 has been proven essential for iron utilization, little is known about it in regulating host immune responses. Here we exhibited that PPE36 was preferentially enriched in Mtb virulent strains and could efficiently inhibit host inflammatory responses and increase bacterial loads in infected macrophages and mice. In exploring the underlying mechanisms, we found that PPE36 could robustly inhibit the activation of inflammatory NF-κB and MAPK (Erk, p38, and Jnk) pathways by promoting E3 ligase Smurf1-mediated ubiquitination and proteasomal degradation of MyD88 protein. Our research revealed a previously unknown function of PPE36 on modulating host immune responses and provided some clues to the development of novel tuberculosis treatment strategies based on immune regulation.

Rv3737 is required for Mycobacterium tuberculosis growth in vitro and in vivo and correlates with bacterial load and disease severity in human tuberculosis.

BACKGROUND: Rv3737 is the sole homologue of multifunctional transporter ThrE in Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether this transporter participates in vitro and in vivo survival of Mtb. METHODS: To characterize the role of Rv3737, we constructed and characterized a Mtb H37RvΔRv3737. This strain was evaluated for altered growth rate and macrophage survival using a cell model of infection. In addition, the comparative analysis was conducted to determine the association between Rv3737 mRNA expression and disease severity in active pulmonary TB patients. RESULTS: The H37RvΔRv3737 strain exhibited significantly slow growth rate compared to H37Rv-WT strain in standard culture medium. Additionally, the survival rate of H37Rv-WT strain in macrophages was 2 folds higher than that of H37RvΔRv3737 at 72 h. A significantly higher level of TNF-α and IL-6 mRNA expression was observed in macrophages infected with H37RvΔRv3737 as compared to H37Rv-WT. Of note, Rv3737 expression was significantly increased in clinical Mtb isolates than H37Rv-WT. The relative expression level of Rv3737 was positively correlated with lung cavity number of TB patients. Similarly, the higher Rv3737 mRNA level resulted in lower C(t) value by Xpert MTB/RIF assay, demonstrating that a positive correlation between Rv3737 expression and bacterial load in TB patients. CONCLUSIONS: Our data takes the lead in demonstrate that the threonine transporter Rv3737 is required for in vitro growth and survival of bacteria inside macrophages. In addition, the expression level of Rv3737 may be associated with bacterial load and disease severity in pulmonary tuberculosis patients.

The effect of rigid cells on blood viscosity: linking rheology and sickle cell anemia.

Sickle cell anemia (SCA) is a disease that affects red blood cells (RBCs). Healthy RBCs are highly deformable objects that under flow can penetrate blood capillaries smaller than their typical size. In SCA there is an impaired deformability of some cells, which are much stiffer and with a different shape than healthy cells, and thereby affect regular blood flow. It is known that blood from patients with SCA has a higher viscosity than normal blood. However, it is unclear how the rigidity of cells is related to the viscosity of blood, in part because SCA patients are often treated with transfusions of variable amounts of normal RBCs and only a fraction of cells will be stiff. Here, we report systematic experimental measurements of the viscosity of a suspension varying the fraction of rigid particles within a suspension of healthy cells. We also perform systematic numerical simulations of a similar mixed suspension of soft RBCs, rigid particles, and their hydrodynamic interactions. Our results show that there is a rheological signature within blood viscosity to clearly identify the fraction of rigidified cells among healthy deformable cells down to a 5% volume fraction of rigidified cells. Although aggregation of RBCs is known to affect blood rheology at low shear rates, and our simulations mimic this effect via an adhesion potential, we show that such adhesion, or aggregation, is unlikely to provide a physical rationalization for the viscosity increase observed in the experiments at moderate shear rates due to rigidified cells. Through numerical simulations, we also highlight that most of the viscosity increase of the suspension is due to the rigidity of the particles rather than their sickled or spherical shape. Our results are relevant to better characterize SCA, provide useful insights relevant to rheological consequences of blood transfusions, and, more generally, extend to the rheology of mixed suspensions having particles with different rigidities, as well as offering possibilities for developments in the field of soft material composites.

Upward trends in new, rifampicin-resistant and concurrent extrapulmonary tuberculosis cases in northern Guizhou Province of China.

Similar to global trends, the incidence rate of tuberculosis (TB) in China declined from 2000 to 2018. In this study, we aimed to evaluate TB trends in northern Guizhou Province and identify risk factors associated with rifampicin-resistant (RR) and concurrent extrapulmonary TB (EPTB). We analyzed data of TB patients hospitalized in Affiliated Hospital of Zunyi Medical University from 2011 to 2018, and assessed correlations between demographic characteristics of patients and RR-TB as well as concurrent EPTB. Our results showed that numbers of new, retreated, RR-TB and concurrent EPTB cases increased gradually from 2011 to 2018. Retreated patients had the highest odds of RR-TB but a lower likelihood of concurrent EPTB compared to new patients. Patients between 21 and 40 years of age had a higher likelihood of RR-TB compared to those 20 years and younger. Female patients and patients from Bijie city as well as the Miao ethnic minority had higher odds of concurrent EPTB. In summary, our data demonstrate upward trends in new, rifampicin-resistant and concurrent extrapulmonary TB cases in northern Guizhou Province of China, which should not be overlooked especially during and post the COVID-19 pandemic because TB is a greater long-term global health threat than COVID-19.

Primary cilium: a paradigm for integrating mathematical modeling with experiments and numerical simulations in mechanobiology.

Primary cilia are non-motile, solitary (one per cell) microtubule-based organelles that emerge from the mother centriole after cells have exited the mitotic cycle. Identified as a mechanosensing organelle that responds to both mechanical and chemical stimuli, the primary cilium provides a fertile ground for integrative investigations of mathematical modeling, numerical simulations, and experiments. Recent experimental findings revealed considerable complexity to the underlying mechanosensory mechanisms that transmit extracellular stimuli to intracellular signaling many of which include primary cilia. In this invited review, we provide a brief survey of experimental findings on primary cilia and how these results lead to various mathematical models of the mechanics of the primary cilium bent under an external forcing such as a fluid flow or a trap. Mathematical modeling of the primary cilium as a fluid-structure interaction problem highlights the importance of basal anchorage and the anisotropic moduli of the microtubules. As theoretical modeling and numerical simulations progress, along with improved state-of-the-art experiments on primary cilia, we hope that details of ciliary regulated mechano-chemical signaling dynamics in cellular physiology will be understood in the near future.

Nanoparticles Incorporating a Fluorescence Turn-on Reporter for Real-Time Drug Release Monitoring, a Chemoenhancer and a Stealth Agent: Poseidon's Trident against Cancer?

The rate and extent of drug release under physiological conditions is a key factor influencing the therapeutic activity of a formulation. Real-time detection of drug release by conventional pharmacokinetics approaches is confounded by low sensitivity, particularly in the case of tissue-targeted novel drug delivery systems, where low concentrations of the drug reach systemic circulation. We present a novel fluorescence turn-on platform for real-time monitoring of drug release from nanoparticles based on reversible fluorescence quenching in fluorescein esters. Fluorescein-conjugated carbon nanotubes (CNTs) were esterified with methotrexate in solution and solid phase, followed by supramolecular functionalization with a chemoenhancer (suramin) or/and a stealth agent (dextran sulfate). Suramin was found to increase the cytotoxicity of methotrexate in A549 cells. On the other hand, dextran sulfate exhibited no effect on cytotoxicity or cellular uptake of CNTs by A549 cells, while a decrease in cellular uptake of CNTs and cytotoxicity of methotrexate was observed in macrophages (RAW 264.7 cells). Similar results were also obtained when CNTs were replaced with graphene. Docking studies revealed that the conjugates are not internalized by folate receptors/transporters. Further, docking and molecular dynamics studies revealed the conjugates do not exhibit affinity toward the methotrexate target, dihydrofolate reductase. Molecular dynamics studies also revealed that distinct features of dextran-CNT and suramin-CNT interactions, characterized by π-π interactions between CNTs and dextran/suramin. Our study provides a simple, cost-effective, and scalable method for the synthesis of nanoparticles conferred with the ability to monitor drug release in real-time. This method could also be extended to other drugs and other types of nanoparticles.

Membrane skeleton modulates erythroid proteome remodeling and organelle clearance.

The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.