Gamblers: An Antibiotic-Induced Evolvable Cell Subpopulation Differentiated by Reactive-Oxygen-Induced General Stress Response.

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.

Robots as models of evolving systems.

Experimental robobiological physics can bring insights into biological evolution. We present a development of hybrid analog/digital autonomous robots with mutable diploid dominant/recessive 6-byte genomes. The robots are capable of death, rebirth, and breeding. We map the quasi-steady-state surviving local density of the robots onto a multidimensional abstract "survival landscape." We show that robot death in complex, self-adaptive stress landscapes proceeds by a general lowering of the robotic genetic diversity, and that stochastically changing landscapes are the most difficult to survive.

Compost incorporation and wildflowers introduction for stormwater infiltration and erosion-control vegetation cover establishment in post-construction landscapes.

Urban and suburban development frequently disturbs and compacts soils, reducing infiltration rates and fertility, posing challenges for post-development vegetation establishment, and contributing to soil erosion. This study investigated the effectiveness of compost incorporation in enhancing stormwater infiltration and vegetation establishment in urban landscapes. Experimental treatments comprised a split-split plot design of vegetation mix (grass, wildflowers, and grass-wildflowers) as main plot, ground cover (hydro-mulch and excelsior) as subplot, and compost (30% Compost and No-Compost) as sub-subplot factors. Wildflower inclusion was motivated by their recognized ecological benefits, including aesthetics, pollinator habitat, and deep root systems. Vegetation cover was assessed using RGB (Red-Green-Blue) imagery and ArcGIS-based supervised image classification. Over a 24-month period, bulk density, infiltration rate, soil penetration resistance, vegetation cover, and root mass density were assessed. Results highlighted that Compost treatments consistently reduced bulk density by 19-24%, lowered soil penetration resistance to under 2 MPa at both field-capacity and water-stressed conditions, and increased infiltration rate by 2-3 times compared to No-Compost treatments. Vegetation cover assessment revealed rapid establishment with 30% compost and 60:40 grass-wildflower mix, persisting for an initial 12 months. Subsequently, all treatments exhibited similar vegetation coverage from 13 to 24 months, reaching 95-100% cover. Compost treatments had significantly higher root mass density within the top 15 cm than No-Compost, but compost addition did not alter the root profile beyond the 15 cm depth incorporation depth. The findings suggest that incorporating 30% compost and including a wildflower or grass-wildflower mix appears to be effective in enhancing stormwater infiltration and provides rapid erosion control vegetation cover establishment in post-construction landscapes.

Model-Free Measurement of Local Entropy Production and Extractable Work in Active Matter.

Time-reversal symmetry breaking and entropy production are universal features of nonequilibrium phenomena. Despite its importance in the physics of active and living systems, the entropy production of systems with many degrees of freedom has remained of little practical significance because the high dimensionality of their state space makes it difficult to measure. Here we introduce a local measure of entropy production and a numerical protocol to estimate it. We establish a connection between the entropy production and extractability of work in a given region of the system and show how this quantity depends crucially on the degrees of freedom being tracked. We validate our approach in theory, simulation, and experiments by considering systems of active Brownian particles undergoing motility-induced phase separation, as well as active Brownian particles and E.coli in a rectifying device in which the time-reversal asymmetry of the particle dynamics couples to spatial asymmetry to reveal its effects on a macroscopic scale.

Emergent Field-Driven Robot Swarm States.

We present an ecology-inspired form of active matter consisting of a robot swarm. Each robot moves over a planar dynamic resource environment represented by a large light-emitting diode array in search of maximum light intensity; the robots deplete (dim) locally by their presence the local light intensity and seek maximum light intensity. Their movement is directed along the steepest local light intensity gradient; we call this emergent symmetry breaking motion "field drive." We show there emerge dynamic and spatial transitions similar to gas, crystalline, liquid, glass, and jammed states as a function of robot density, resource consumption rates, and resource recovery rates. Paradoxically the nongas states emerge from smooth, flat resource landscapes, not rough ones, and each state can directly move to a glassy state if the resource recovery rate is slow enough, at any robot density.

Emergence of Escherichia coli critically buckled motile helices under stress.

Bacteria under external stress can reveal unexpected emergent phenotypes. We show that the intensely studied bacterium Escherichia coli can transform into long, highly motile helical filaments poized at a torsional buckling criticality when exposed to minimum inhibitory concentrations of several antibiotics. While the highly motile helices are physically either right- or left-handed, the motile helices always rotate with a right-handed angular velocity [Formula: see text], which points in the same direction as the translational velocity [Formula: see text] of the helix. Furthermore, these helical cells do not swim by a "run and tumble" but rather synchronously flip their spin [Formula: see text] and thus translational velocity-backing up rather than tumbling. By increasing the translational persistence length, these dynamics give rise to an effective diffusion coefficient up to 20 times that of a normal E. coli cell. Finally, we propose an evolutionary mechanism for this phenotype's emergence whereby the increased effective diffusivity provides a fitness advantage in allowing filamentous cells to more readily escape regions of high external stress.

It doesn't always pay to be fit: success landscapes.

Landscapes play an important role in many areas of biology, in which biological lives are deeply entangled. Here we discuss a form of landscape in evolutionary biology which takes into account (1) initial growth rates, (2) mutation rates, (3) resource consumption by organisms, and (4) cyclic changes in the resources with time. The long-term equilibrium number of surviving organisms as a function of these four parameters forms what we call a success landscape, a landscape we would claim is qualitatively different from fitness landscapes which commonly do not include mutations or resource consumption/changes in mapping genomes to the final number of survivors. Although our analysis is purely theoretical, we believe the results have possibly strong connections to how we might treat diseases such as cancer in the future with a deeper understanding of the interplay between resource degradation, mutation, and uncontrolled cell growth.

Broken flow symmetry explains the dynamics of small particles in deterministic lateral displacement arrays.

Deterministic lateral displacement (DLD) is a technique for size fractionation of particles in continuous flow that has shown great potential for biological applications. Several theoretical models have been proposed, but experimental evidence has demonstrated that a rich class of intermediate migration behavior exists, which is not predicted. We present a unified theoretical framework to infer the path of particles in the whole array on the basis of trajectories in a unit cell. This framework explains many of the unexpected particle trajectories reported and can be used to design arrays for even nanoscale particle fractionation. We performed experiments that verify these predictions and used our model to develop a condenser array that achieves full particle separation with a single fluidic input.

Cervical Stenosis: Previously Unrecognized Cause of False-Negative Human Papillomavirus Tests in Women Developing Cervical Cancer.

OBJECTIVES: Cervical stenosis can jeopardize adequate posttreatment cytologic follow-up of patients treated for high-grade cervical intraepithelial lesions. An impact on human papillomavirus (HPV) testing has not been described. MATERIALS AND METHODS: We describe 2 patients with cervical stenosis, followed by cytology and HPV co-testing after excisions of high-grade cervical intraepithelial lesions. Each had 1 or more co-test "double-negative" results. Hysterectomies revealed unexpected cervical carcinomas. RESULTS: In case 1, an 80-year-old woman with complete cervical stenosis and earlier high-grade squamous dysplasia presented with abdominal pain, nausea, and an enlarged uterus. Attempted endometrial biopsy was unsuccessful. Cytology and HPV tests 9 months earlier were negative. Hysterectomy revealed a cervical squamous carcinoma. In case 2, a 40-year-old woman followed conservatively after excision of endocervical adenocarcinoma in situ had 5 follow-up cytology and HPV co-tests. All were HPV negative. Elective hysterectomy revealed cervical adenocarcinoma. Both carcinomas tested HPV positive. CONCLUSIONS: Cervical stenosis in women developing cervical cancer can cause misleading sampling and false-negative HPV test results.

Polyploid giant cancer cells: Unrecognized actuators of tumorigenesis, metastasis, and resistance.

Cancer led to the deaths of more than 9 million people worldwide in 2018, and most of these deaths were due to metastatic tumor burden. While in most cases, we still do not know why cancer is lethal, we know that a total tumor burden of 1 kg-equivalent to one trillion cells-is not compatible with life. While localized disease is curable through surgical removal or radiation, once cancer has spread, it is largely incurable. The inability to cure metastatic cancer lies, at least in part, to the fact that cancer is resistant to all known compounds and anticancer drugs. The source of this resistance remains undefined. In fact, the vast majority of metastatic cancers are resistant to all currently available anticancer therapies, including chemotherapy, hormone therapy, immunotherapy, and systemic radiation. Thus, despite decades-even centuries-of research, metastatic cancer remains lethal and incurable. We present historical and contemporary evidence that the key actuators of this process-of tumorigenesis, metastasis, and therapy resistance-are polyploid giant cancer cells.

Oriented collagen fibers direct tumor cell intravasation.

In this work, we constructed a Collagen I-Matrigel composite extracellular matrix (ECM). The composite ECM was used to determine the influence of the local collagen fiber orientation on the collective intravasation ability of tumor cells. We found that the local fiber alignment enhanced cell-ECM interactions. Specifically, metastatic MDA-MB-231 breast cancer cells followed the local fiber alignment direction during the intravasation into rigid Matrigel (∼10 mg/mL protein concentration).

Rapid emergence and mechanisms of resistance by U87 glioblastoma cells to doxorubicin in an in vitro tumor microfluidic ecology.

In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges of drug and nutrient gradients and local populations. This ecology of a fragmented metapopulation induced the drug resistance in stage IV U87 glioblastoma cells to doxorubicin in 7 d. Exome and transcriptome sequencing of the resistant cells identified mutations and differentially expressed genes. Gene ontology and pathway analyses of the genes identified showed that they were functionally relevant to the established mechanisms of doxorubicin action. Specifically, we identified (i) a frame-shift insertion in the filamin-A gene, which regulates the influx and efflux of topoisomerase II poisons; (ii) the overexpression of aldo-keto reductase enzymes, which convert doxorubicin into doxorubicinol; and (iii) activation of NF-κB via alterations in the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway from mutations in three genes (CARD6, NSD1, and NLRP13) and the overexpression of inflammatory cytokines. Functional experiments support the in silico analyses and, together, demonstrate the effects of these genetic changes. Our findings suggest that, given the rapid evolution of resistance and the focused response, this technology could act as a rapid screening modality for genetic aberrations leading to resistance to chemotherapy as well as counter selection of drugs unlikely to be successful ultimately.

Emergence of antibiotic resistance from multinucleated bacterial filaments.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

Ancient hot and cold genes and chemotherapy resistance emergence.

We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.

Automated leukocyte processing by microfluidic deterministic lateral displacement.

We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples. © 2016 International Society for Advancement of Cytometry.

Soil Weathering as an Engine for Manganese Contamination of Well Water.

Manganese (Mn) contamination of well water is recognized as an environmental health concern. In the southeastern Piedmont region of the United States, well water Mn concentrations can be >2 orders of magnitude above health limits, but the specific sources and causes of elevated Mn in groundwater are generally unknown. Here, using field, laboratory, spectroscopic, and geospatial analyses, we propose that natural pedogenetic and hydrogeochemical processes couple to export Mn from the near-surface to fractured-bedrock aquifers within the Piedmont. Dissolved Mn concentrations are greatest just below the water table and decrease with depth. Solid-phase concentration, chemical extraction, and X-ray absorption spectroscopy data show that secondary Mn oxides accumulate near the water table within the chemically weathering saprolite, whereas less-reactive, primary Mn-bearing minerals dominate Mn speciation within the physically weathered transition zone and bedrock. Mass-balance calculations indicate soil weathering has depleted over 40% of the original solid-phase Mn from the near-surface, and hydrologic gradients provide a driving force for downward delivery of Mn. Overall, we estimate that >1 million people in the southeastern Piedmont consume well water containing Mn at concentrations exceeding recommended standards, and collectively, these results suggest that integrated soil-bedrock-system analyses are needed to predict and manage Mn in drinking-water wells.

Cell motility and drug gradients in the emergence of resistance to chemotherapy.

The emergence of resistance to chemotherapy by cancer cells, when combined with metastasis, is the primary driver of mortality in cancer and has proven to be refractory to many efforts. Theory and computer modeling suggest that the rate of emergence of resistance is driven by the strong selective pressure of mutagenic chemotherapy and enhanced by the motility of mutant cells in a chemotherapy gradient to areas of higher drug concentration and lower population competition. To test these models, we constructed a synthetic microecology which superposed a mutagenic doxorubicin gradient across a population of motile, metastatic breast cancer cells (MDA-MB-231). We observed the emergence of MDA-MB-231 cancer cells capable of proliferation at 200 nM doxorubicin in this complex microecology. Individual cell tracking showed both movement of the MDA-MB-231 cancer cells toward higher drug concentrations and proliferation of the cells at the highest doxorubicin concentrations within 72 h, showing the importance of both motility and drug gradients in the emergence of resistance.

Minimization of thermodynamic costs in cancer cell invasion.

Metastasis, the truly lethal aspect of cancer, occurs when metastatic cancer cells in a tumor break through the basement membrane and penetrate the extracellular matrix. We show that MDA-MB-231 metastatic breast cancer cells cooperatively invade a 3D collagen matrix while following a glucose gradient. The invasion front of the cells is a dynamic one, with different cells assuming the lead on a time scale of 70 h. The front cell leadership is dynamic presumably because of metabolic costs associated with a long-range strain field that precedes the invading cell front, which we have imaged using confocal imaging and marker beads imbedded in the collagen matrix. We suggest this could be a quantitative assay for an invasive phenotype tracking a glucose gradient and show that the invading cells act in a cooperative manner by exchanging leaders in the invading front.