•Direct numerical simulation (DNS), implementing the level set method, of vertical two-phase flow regime transition from slug flow to churn-turbulent flow in a pipe using the PHASTA code.•Behavior of ...the two-phase flow simulation is shown to be strongly dependent on the pipe length and mesh resolution.•The DNS is shown to be in good agreement with experiments and theory.•The simulations show the transition from slug to churn flow is related to when the wake of the bubble reaches the nose of the following bubble.
Direct numerical simulation (DNS) coupled with an interface tracking method (ITM) is used to demonstrate the applicability of DNS to the study of vertical two-phase flow regime transition. The study focuses on the slug flow to churn-turbulent regime transition. PHASTA, a finite element based flow solver which utilizes the level set method for interface tracking, has been used to perform the presented simulations. A domain size study has been conducted in order to find pipe dimensions that match the natural wavelength of the periodic slug flow. A mesh resolution study has been completed. The results show that using DNS to simulate the slug-to-churn transition is within the capabilities of the code as the simulations agree well with experimental data and empirical knowledge. The simulations were analyzed to find flow features such as the velocity profile in the wake of a Taylor bubble and the bubble interface shape evolution during breakdown. An understanding of such features could help identify the driving physics behind the transition phenomenon.
Force-sensitive arrest peptides regulate protein biosynthesis by stalling the ribosome as they are translated. Synthesis can be resumed when the nascent arrest peptide experiences a pulling force of ...sufficient magnitude to break the stall. Efficient stalling is dependent on the specific identity of a large number of amino acids, including amino acids that are tens of angstroms away from the peptidyl transferase center (PTC). The mechanism of force-induced restart and the role of these essential amino acids far from the PTC is currently unknown. We use hundreds of independent molecular dynamics trajectories spanning over 120 μs in combination with kinetic analysis to characterize multiple barriers along the force-induced restart pathway for the arrest peptide SecM. We find that the essential amino acids far from the PTC play a major role in controlling the transduction of applied force. In successive states along the stall-breaking pathway, the applied force propagates up the nascent chain until it reaches the C-terminus of SecM and the PTC, inducing conformational changes that allow for restart of translation. A similar mechanism of force propagation through multiple states is observed in the VemP stall-breaking pathway, but secondary structure in VemP allows for heterogeneity in the order of transitions through intermediate states. Results from both arrest peptides explain how residues that are tens of angstroms away from the catalytic center of the ribosome impact stalling efficiency by mediating the response to an applied force and shielding the amino acids responsible for maintaining the stalled state of the PTC.
Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To ...improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.
•Efficient protocol to generate human microglia from PSCs•iPSC-derived microglia have ramified morphology and motile processes•Expression and cytokine profiles of iPSC-derived microglia resemble primary microglia•iPSC-derived microglia can phagocytose beads and respond to ADP
Douvaras and colleagues generated microglia from pluripotent stem cells using a chemically defined protocol through a myeloid progenitor. iPSC-derived microglia showed highly motile processes, were able to phagocytose and responded to ADP with calcium transients. Microglial identity was further confirmed by gene expression analysis comparing multiple iPSC-derived microglia samples with primary microglia and peripheral blood or other tissue-specific macrophages.
Proteolysis targeting chimera (PROTAC) is a novel drug modality that facilitates the degradation of a target protein by inducing proximity with an E3 ligase. In this work, we present a new ...computational framework to model the cooperativity between PROTAC–E3 binding and PROTAC–target binding principally through protein–protein interactions (PPIs) induced by the PROTAC. Due to the scarcity and low resolution of experimental measurements, the physical and chemical drivers of these non-native PPIs remain to be elucidated. We develop a coarse-grained (CG) approach to model interactions in the target–PROTAC–E3 complexes, which enables converged thermodynamic estimations using alchemical free energy calculation methods despite an unconventional scale of perturbations. With minimal parametrization, we successfully capture fundamental principles of cooperativity, including the optimality of intermediate PROTAC linker lengths that originates from configurational entropy. We qualitatively characterize the dependency of cooperativity on PROTAC linker lengths and protein charges and shapes. Minimal inclusion of sequence- and conformation-specific features in our current force field, however, limits quantitative modeling to reproduce experimental measurements, but further development of the CG model may allow for efficient computational screening to optimize PROTAC cooperativity.
New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed ...in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.
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•CD49f is a novel, reactivity-independent marker for human astrocytes•CD49f can be used to purify human fetal astrocytes and iPSC-derived astrocytes•CD49f+ hiPSC-astrocytes acquire an A1-like reactive state upon cytokine stimulation•CD49f+ A1-like reactive astrocytes are dysfunctional and toxic to neurons in vitro
Barbar et al. identify CD49f as a novel surface marker expressed by human astrocytes that can purify hiPSC-astrocytes and primary fetal astrocytes. CD49f+ hiPSC-astrocytes respond to pro-inflammatory stimuli and become A1 reactive astrocytes, which are dysfunctional and secrete neurotoxic factors that induce apoptosis in human and rodent neurons.
Replacement of mitochondria through nuclear transfer between oocytes of two different women has emerged recently as a strategy for preventing inheritance of mtDNA diseases. Although experiments in ...human oocytes have shown effective replacement, the consequences of small amounts of mtDNA carryover have not been studied sufficiently. Using human mitochondrial replacement stem cell lines, we show that, even though the low levels of heteroplasmy introduced into human oocytes by mitochondrial carryover during nuclear transfer often vanish, they can sometimes instead result in mtDNA genotypic drift and reversion to the original genotype. Comparison of cells with identical oocyte-derived nuclear DNA but different mtDNA shows that either mtDNA genotype is compatible with the nucleus and that drift is independent of mitochondrial function. Thus, although functional replacement of the mitochondrial genome is possible, even low levels of heteroplasmy can affect the stability of the mtDNA genotype and compromise the efficacy of mitochondrial replacement.
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•Cryopreservation allows coordinated nuclear transfer in human oocytes•Stem cell lines and derivatives show low-level carryover of transferred mtDNA•Recipient mtDNA is functionally compatible with donor nuclear DNA•Genetic drift can lead to restoration of the original donor mitochondrial genotype
Yamada et al. show, using human cells, that even small amounts of mtDNA carried over during nuclear transfer for mitochondrial replacement can lead to mtDNA genotype reversion. This situation would need to be avoided for clinical application and stable prevention of mtDNA diseases.
Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative ...medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.
Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM ...are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.
Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology that affects the CNS. While current therapies are primarily directed against the immune system, the new challenge is to ...address progressive MS with remyelinating and neuroprotective strategies. Here, we develop a highly reproducible protocol to efficiently derive oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from induced pluripotent stem cells (iPSCs). Key elements of our protocol include adherent cultures, dual SMAD inhibition, and addition of retinoids from the beginning of differentiation, which lead to increased yields of OLIG2 progenitors and high numbers of OPCs within 75 days. Furthermore, we show the generation of viral and integration-free iPSCs from primary progressive MS (PPMS) patients and their efficient differentiation to oligodendrocytes. PPMS OPCs are functional, as demonstrated by in vivo myelination in the shiverer mouse. These results provide encouraging advances toward the development of autologous cell therapies using iPSCs.
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•Development of a rapid, highly efficient, and robust protocol to produce O4+ hOPCs•Generation of iPSCs from primary progressive MS patients•Differentiation of PPMS iPSCs to mature oligodendrocytes in vitro•Successful in vivo myelination from PPMS-iPSC-derived OPCs
In this article, Fossati and colleagues describe an efficient and highly reproducible differentiation protocol to generate oligodendrocyte progenitor cells (OPCs) from induced pluripotent stem cells. Using this protocol, they show that OPCs from primary progressive multiple sclerosis (MS) patients are able to myelinate neurons in the shiverer mouse model, providing the premise for the development of future autologous cell transplantation therapies in MS.
Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms, including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins ...from a single mRNA transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work, we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during its biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamics simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.
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