Suchergebnisse

Abstract
The process of cell differentiation in multicellular organisms is characterized by hierarchy and irreversibility in many cases. However, the conditions and selection pressures that give rise to these characteristics remain poorly understood. By using a mathematical model, here we show that the network of differentiation potency (differentiation diagram) becomes necessarily hierarchical and irreversible by increasing the number of terminally differentiated states under certain conditions. The mechanisms generating these characteristics are clarified using geometry in the cell state space. The results demonstrate that the hierarchical organization and irreversibility can manifest independently of direct selection pressures associated with these characteristics, instead they appear to evolve as byproducts of selective forces favoring a diversity of differentiated cell types. The study also provides a new perspective on the structure of gene regulatory networks that produce hierarchical and irreversible differentiation diagrams. These results indicate some constraints on cell differentiation, which are expected to provide a starting point for theoretical discussion of the implicit limits and directions of evolution in multicellular organisms.

Abstract
All lipid membranes have inherent morphological preferences and resist deformation. Yet adaptations in membrane shape can and do occur at multiple length scales. While this plasticity is crucial for cellular physiology, the factors controlling the morphological energetics of lipid bilayers and the dominant mechanisms of membrane remodeling remain to be fully understood. An ongoing debate regarding the universality of the stiffening effect of cholesterol underscores the challenges facing this field, both experimentally and theoretically, even for simple lipid mixtures. On the computational side, we have argued that enhanced-sampling all-atom molecular dynamics simulations are uniquely suited for the quantification of membrane conformational energetics, as they minimize a priori assumptions and permit analysis of bilayers in deformed states. To showcase this approach, we examine reported inconsistencies between alternative experimental measurements of bending moduli for cholesterol-enriched membranes. Specifically, we analyze lipid bilayers with different chain saturation and compute free-energy landscapes for curvature deformations distributed over areas from ∼5 to ∼60 nm2. These enhanced simulations, totaling over 100 μs of sampling time, enable us to directly quantify both bending and tilt moduli and to dissect the contributing factors and molecular mechanisms of curvature generation at each length scale. Our results show that the effects of cholesterol on bending rigidity are lipid-specific and suggest that this specificity arises from differences in the torsional dynamics of the acyl chains. In summary, we demonstrate that quantitative relationships can now be established between lipid structure and bending energetics, paving the way for addressing open fundamental questions in cell membrane mechanics.

Abstract
Manipulation of protein synthesis is commonly applied to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms have been widely investigated and employed in bio-related research, but it is extraordinarily challenging to measure and evaluate the synthesis inhibition efficiencies of individual proteins by different inhibitors at the proteome level. Newly synthesized proteins are the immediate and direct products of protein synthesis, and thus their comprehensive quantification provides a unique opportunity to study protein inhibition. Here, we systematically investigate protein inhibition and evaluate different popular inhibitors, i.e. cycloheximide, puromycin, and anisomycin, through global quantification of newly synthesized proteins in several types of human cells (A549, MCF-7, Jurkat, and THP-1 cells). The inhibition efficiencies of protein synthesis are comprehensively measured by integrating azidohomoalanine-based protein labeling, selective enrichment, a boosting approach, and multiplexed proteomics. The same inhibitor results in dramatic variation of the synthesis inhibition efficiencies for different proteins in the same cells, and each inhibitor exhibits unique preferences. Besides cell type- and inhibitor-specific effects, some universal rules are unraveled. For instance, nucleolar and ribosomal proteins have relatively higher inhibition efficiencies in every type of cells treated with each inhibitor. Moreover, proteins intrinsically resistant or sensitive to the inhibition are identified and found to have distinct functions. Systematic investigation of protein synthesis inhibition in several types of human cells by different inhibitors provides valuable information about the inhibition of protein synthesis, advancing our understanding of inhibiting protein synthesis.

Abstract
We show that zinc finger imprinted 3 (Zim3), when used as Zim3-KRAB-dCas9 effector in interference CRISPR, without any guide RNAs, paradoxically up-regulates key cardiac ion channel genes in human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), responsible for healthy resting membrane potential, repolarization of the action potential, and electrical transmission of signals. These were found to yield expected functional enhancements consistent with a more mature iPSC-CM phenotype, with potentially desirable properties.

Abstract
Phagocytic elimination of solid tumors by innate immune cells seems attractive for immunotherapy, particularly because of the possibilities for acquired immunity. However, the approach remains challenging, with blockade of the macrophage checkpoint CD47 working in immunodeficient mice and against highly immunogenic tumors but not in the clinic where tumors are poorly immunogenic. Even when mouse tumors of poorly immunogenic B16F10 melanoma are opsonized to drive engulfment with a suitable monoclonal antibody (mAb), anti-CD47 blockade remains insufficient. Using both in vitro immuno-tumoroids and in vivo mouse models, we show with CRISPR interference (CRISPRi) that a relatively uniform minimum repression of CD47 by 80% is needed for phagocytosis to dominate net growth when combined with an otherwise ineffective mAb (anti-Tyrp1). Heterogeneity enriches for CD47-high cells, but mice that eliminate tumors generate prophagocytic IgGs that increase in titer with CD47 repression and with tumor accumulation of macrophages, although deeper repression does not improve survival. Given well-known limitations of antibody permeation into solid tumors, our studies clarify benchmarks for CD47 disruption that should be more clinically feasible and safer but just as effective as complete ablation. Additionally, safe but ineffective opsonization in human melanoma trials suggests that combinations with deep repression of CD47 could prove effective and initiate durable immunity.

Abstract
The ability of cells to sense and adapt to curvy topographical features has been implicated in organ morphogenesis, tissue repair, and tumor metastasis. However, how individual cells or multicellular assemblies sense and differentiate curvatures remains elusive. Here, we reveal a curvature sensing mechanism in which surface tension can selectively activate either actin or integrin flows, leading to bifurcating cell migration modes: focal adhesion formation that enables cell crawling at convex front edges and actin cable assembly that pulls cells forward at concave front edges. The molecular flows and curved front morphogenesis are sustained by coordinated cellular tension generation and transmission. We track the molecular flows and mechanical force transduction pathways by a phase-field model, which predicts that multicellular curvature sensing is more efficient than individual cells, suggesting collective intelligence of cells. The unique ability of cells in curvature sensing and migration mode bifurcating may offer insights into emergent collective patterns and functions of living active systems at different length scales.

Abstract
Legionnaires' disease (LD) is a severe form of pneumonia (∼10–25% fatality rate) caused by inhalation of aerosols containing Legionella, a pathogenic gram-negative bacteria. These bacteria can grow, spread, and aerosolize through building water systems. A recent dramatic increase in LD incidence has been observed globally, with a 9-fold increase in the United States from 2000 to 2018, and with disproportionately higher burden for socioeconomically vulnerable subgroups. Despite the focus of decades of research since the infamous 1976 outbreak, substantial knowledge gaps remain with regard to source of exposure and the reason(s) for the dramatic increase in LD incidence. Here, we rule out factors indicated in literature to contribute to its long-term increases and identify a hitherto unexplored explanatory factor. We also provide an epidemiological demonstration that the occurrence of LD is linked with exposure to cooling towers (CTs). Our results suggest that declining sulfur dioxide air pollution, which has many well-established health benefits, results in reduced acidity of aerosols emitted from CTs, which may prolong the survival duration of Legionella in contaminated CT droplets and contribute to the increase in LD incidence. Mechanistically associating decreasing aerosol acidity with this respiratory disease has implications for better understanding its transmission, predicting future risks, and informed design of preventive and interventional strategies that consider the complex impacts of continued sulfur dioxide changes.

Abstract
Red blood cell (RBC) aging manifests through progressive changes in cell morphology, rigidity, and expression of membrane proteins. To maintain the quality of circulating blood, splenic macrophages detect the biochemical signals and biophysical changes of RBCs and selectively clear them through erythrophagocytosis. In sickle cell disease (SCD), RBCs display alterations affecting their interaction with macrophages, leading to aberrant phagocytosis that may cause life-threatening spleen sequestration crises. To illuminate the mechanistic control of RBC engulfment by macrophages in SCD, we integrate a system biology model of RBC-macrophage signaling interactions with a biophysical model of macrophage engulfment, as well as in vitro phagocytosis experiments using the spleen-on-a-chip technology. Our modeling framework accurately predicts the phagocytosis dynamics of RBCs under different disease conditions, reveals patterns distinguishing normal and sickle RBCs, and identifies molecular targets including Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP1) and cluster of differentiation 47 (CD47)/signal regulatory protein α (SIRPα) as therapeutic targets to facilitate the controlled clearance of sickle RBCs in the spleen.

Abstract
Research in human volunteers and surgical patients has shown that unconsciousness under general anesthesia can be reliably tracked using real-time electroencephalogram processing. Hence, a closed-loop anesthesia delivery (CLAD) system that maintains precisely specified levels of unconsciousness is feasible and would greatly aid intraoperative patient management. The US Federal Drug Administration has approved no CLAD system for human use due partly to a lack of testing in appropriate animal models. To address this key roadblock, we implement a nonhuman primate (NHP) CLAD system that controls the level of unconsciousness using the anesthetic propofol. The key system components are a local field potential (LFP) recording system; propofol pharmacokinetics and pharmacodynamic models; the control variable (LFP power between 20 and 30 Hz), a programmable infusion system and a linear quadratic integral controller. Our CLAD system accurately controlled the level of unconsciousness along two different 125-min dynamic target trajectories for 18 h and 45 min in nine experiments in two NHPs. System performance measures were comparable or superior to those in previous CLAD reports. We demonstrate that an NHP CLAD system can reliably and accurately control in real-time unconsciousness maintained by anesthesia. Our findings establish critical steps for CLAD systems' design and testing prior to human testing.

Abstract
Particulate matter (PM) is a ubiquitous component of air pollution that is epidemiologically linked to human pulmonary diseases. PM chemical composition varies widely, and the development of high-throughput experimental techniques enables direct profiling of cellular effects using compositionally unique PM mixtures. Here, we show that in a human bronchial epithelial cell model, exposure to three chemically distinct PM mixtures drive unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability, DNA damage responses, and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Finally, we observed that PM mixtures with higher cadmium content induced increased DNA damage and drove redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of individual cellular morphologies provides a robust, high-throughput approach to gauge the effects of environmental stressors on biological systems and score cellular susceptibilities to pollution.

AbstractCyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

Abstract
Chronic and genetic kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD) have few therapeutic options, and clinical trials testing small molecule drugs have been unfavorable due to low kidney bioavailability and adverse side effects. Although nanoparticles can be designed to deliver drugs directly to the diseased site, there are no kidney-targeted nanomedicines clinically available, and most FDA-approved nanoparticles are administered intravenously which is not ideal for chronic diseases. To meet these challenges of chronic diseases, we developed a biomaterials-based strategy using chitosan particles (CP) for oral delivery of therapeutic, kidney-targeting peptide amphiphile micelles (KMs). We hypothesized that encapsuling KMs into CP would enhance the bioavailability of KMs upon oral administration given the high stability of chitosan in acidic conditions and mucoadhesive properties enabling absorption within the intestines. To test this, we evaluated the mechanism of KM access to the kidneys via intravital imaging and investigated the KM biodistribution in a porcine model. Next, we loaded KMs carrying the ADPKD drug metformin into CP (KM-CP-met) and measured in vitro therapeutic effect. Upon oral administration in vivo, KM-CP-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM only or free drug. As such, KM-CP-met treatment in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre which develops the disease over 120 days and mimics the slow development of ADPKD) showed enhanced therapeutic efficacy without affecting safety despite repeated treatment. Herein, we demonstrate the potential of KM-CP as a nanomedicine strategy for oral delivery for the long-term treatment of chronic kidney diseases.

Abstract
Understanding the biomechanical behavior of the intervertebral disc is crucial for studying disease mechanisms and developing tissue engineering strategies for managing disc degeneration. We used synchrotron small-angle X-ray scattering to investigate how changes to collagen behavior contribute to alterations in the disc's ability to resist compression. Coccygeal motion segments from 6-month-old lean Sprague-Dawley rats ( n=7) and diabetic obese University of California Davis type 2 diabetes mellitus (UCD-T2DM) rats ( n=6, diabetic for 68±7 days) were compressed during simultaneous synchrotron scanning to measure collagen strain at the nanoscale (beamline 7.3.3 of the Advanced Light Source). After compression, the annulus fibrosus was assayed for nonenzymatic cross-links. In discs from lean rats, resistance to compression involved two main energy-dissipation mechanisms at the nanoscale: (1) rotation of the two groups of collagen fibrils forming the annulus fibrosus and (2) straightening (uncrimping) and stretching of the collagen fibrils. In discs from diabetic rats, both mechanisms were significantly impaired. Specifically, diabetes reduced fibril rotation by 31% and reduced collagen fibril strain by 30% (compared to lean discs). The stiffening of collagen fibrils in the discs from diabetic rats was consistent with a 31% higher concentration of nonenzymatic cross-links and with evidence of earlier onset plastic deformations such as fibril sliding and fibril–matrix delamination. These findings suggest that fibril reorientation, stretching, and straightening are key deformation mechanisms that facilitate whole-disc compression, and that type 2 diabetes impairs these efficient and low-energy elastic deformation mechanisms, thereby altering whole-disc behavior and inducing the earlier onset of plastic deformation.

Abstract
The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-μm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Abstract
Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.