Diversity and Composition of Methanotroph Communities in Caves.

Methane oxidizing microorganisms (methanotrophs) are ubiquitous in the environment and represent a major sink for the greenhouse gas methane (CH4). Recent studies have demonstrated methanotrophs are abundant and contribute to CH4 dynamics in caves. However, very little is known about what controls the distribution and abundance of methanotrophs in subterranean ecosystems. Here, we report a survey of soils collected from > 20 caves in North America to elucidate the factors shaping cave methanotroph communities. Using 16S rRNA sequencing, we recovered methanotrophs from nearly all (98%) of the samples, including cave sites where CH4 concentrations were at or below detection limits (≤0.3 ppmv). We identified a core methanotroph community among caves comprised of high-affinity methanotrophs. Although associated with local-scale mineralogy, methanotroph composition did not systematically vary between the entrances and interior of caves, where CH4 concentrations varied. We also observed methanotrophs are able to disperse readily between cave systems showing these organisms have low barriers to dispersal. Lastly, the relative abundance of methanotrophs was positively correlated with cave-air CH4 concentrations, suggesting these microorganisms contribute to CH4 flux in subterranean ecosystems. IMPORTANCE Recent observations have shown the atmospheric greenhouse gas methane (CH4) is consumed by microorganisms (methanotrophs) in caves at rates comparable to CH4 oxidation in surface soils. Caves are abundant in karst landscapes that comprise 14% of Earth's land surface area, and therefore may represent a potentially important, but overlooked, CH4 sink. We sampled cave soils to gain a better understand the community composition and structure of cave methanotrophs. Our results show the members of the USC-γ clade are dominant in cave communities and can easily disperse through the environment, methanotroph relative abundance was correlated with local scale mineralogy of soils, and the relative abundance of methanotrophs was positively correlated with CH4 concentrations in cave air.

Registration Attendants Show Poor Readiness to Handle Advanced Care Planning Discussions.

Background: Emergency departments (ED) and other medical points of care are required to provide patients with advance directive (AD) information. Although many hospitals provide AD information in EDs, the comfort and preparation of the ED staff with this responsibility is unclear. Objective: To determine the attitudes, comfort levels, and prior training of ED staff with AD. Methods: The ED social workers, nurses, registration attendants, residents, and attending physicians at two academic hospitals completed a survey about their attitudes around, preparedness for, and experiences with advance care planning (ACP) discussions in the ED. Results: We received responses from 220 ED staff. Preparedness to discuss ACP with patients varied by profession. Eighty percent of social workers (n = 4/5) and 52% (n = 16/31) of attending physicians reported preparedness to handle ACP discussions. Registration attendants were the least prepared, and only 4% (n = 1/24) reported preparedness to discuss ACP. Attempts at ACP discussions with patients also differed by profession, with attending physicians being the most likely (77%, n = 24/31), whereas registration attendants were the least likely (8%, n = 2/24). Fifty-nine percent of surveyed staff (n = 130/220) believed that ACP was a component of emergency care, although only 13% (n = 29/220) had received training. Conclusion: The ED staff are in favor of ACP in the ED. Preparedness for, and attempts of ACP discussions with patients in the ED vary by profession. Attending physicians and social workers tend to be the most prepared, and they report the most frequent attempts at discussions with patients. Despite the fact that registration attendants are frequently tasked with asking about patient ADs, they show little confidence in asking about and discussing such matters. Our research indicates that registration attendants feel unprepared to guide discussions of ADs and should not do so without additional training.

Rainforest-to-pasture conversion stimulates soil methanogenesis across the Brazilian Amazon.

The Amazon rainforest is a biodiversity hotspot and large terrestrial carbon sink threatened by agricultural conversion. Rainforest-to-pasture conversion stimulates the release of methane, a potent greenhouse gas. The biotic methane cycle is driven by microorganisms; therefore, this study focused on active methane-cycling microorganisms and their functions across land-use types. We collected intact soil cores from three land use types (primary rainforest, pasture, and secondary rainforest) of two geographically distinct areas of the Brazilian Amazon (Santarém, Pará and Ariquemes, Rondônia) and performed DNA stable-isotope probing coupled with metagenomics to identify the active methanotrophs and methanogens. At both locations, we observed a significant change in the composition of the isotope-labeled methane-cycling microbial community across land use types, specifically an increase in the abundance and diversity of active methanogens in pastures. We conclude that a significant increase in the abundance and activity of methanogens in pasture soils could drive increased soil methane emissions. Furthermore, we found that secondary rainforests had decreased methanogenic activity similar to primary rainforests, and thus a potential to recover as methane sinks, making it conceivable for forest restoration to offset greenhouse gas emissions in the tropics. These findings are critical for informing land management practices and global tropical rainforest conservation.

The Oldest Highlands of Mars May Be Massive Dust Fallout Deposits.

The oldest terrains of Mars are cratered landscapes, in which extensive valleys and basins are covered by ubiquitous fluvial plains. One current paradigm maintains that an impact-generated megaregolith underlies these sediments. This megaregolith was likely largely generated during the Early Noachian (~4.1 to ~3.94 Ga) when most Martian impact basins formed. We examined the geologic records of NW Hellas and NW Isidis, which include this epoch's most extensive circum-basin outcrops. Here, we show that these regions include widespread, wind-eroded landscapes, crater rims eroded down by several hundred meters, pitted plains, and inverted fluvial and crater landforms. These surfaces exhibit few fresh craters, indicating geologically recent wind erosion. The deep erosion, topographic inversions, and an absence of dunes on or near talus across these regions suggest that sediments finer than sand compose most of these highland materials. We propose that basin-impact-generated hurricane-force winds created sediment-laden atmospheric conditions, and that muddy rains rapidly settled suspended sediments to construct extensive Early Noachian highlands. The implied high abundance of fine-grained sediments before these impacts suggests large-scale glacial silt production and supports the previously proposed Noachian "icy highlands" hypothesis. We suggest that subglacial meltwater interactions with the sedimentary highlands could have promoted habitability, particularly in clay strata.

The Chaotic Terrains of Mercury Reveal a History of Planetary Volatile Retention and Loss in the Innermost Solar System.

Mercury's images obtained by the 1974 Mariner 10 flybys show extensive cratered landscapes degraded into vast knob fields, known as chaotic terrain (AKA hilly and lineated terrain). For nearly half a century, it was considered that these terrains formed due to catastrophic quakes and ejecta fallout produced by the antipodal Caloris basin impact. Here, we present the terrains' first geologic examination based on higher spatial resolution MESSENGER (MErcury Surface Space ENvironment GEochemistry and Ranging) imagery and laser altimeter topography. Our surface age determinations indicate that their development persisted until ~1.8 Ga, or ~2 Gyrs after the Caloris basin formed. Furthermore, we identified multiple chaotic terrains with no antipodal impact basins; hence a new geological explanation is needed. Our examination of the Caloris basin's antipodal chaotic terrain reveals multi-kilometer surface elevation losses and widespread landform retention, indicating an origin due to major, gradual collapse of a volatile-rich layer. Crater interior plains, possibly lavas, share the chaotic terrains' age, suggesting a development associated with a geothermal disturbance above intrusive magma bodies, which best explains their regionality and the enormity of the apparent volume losses involved in their development. Furthermore, evidence of localized, surficial collapse, might reflect a complementary, and perhaps longer lasting, devolatilization history by solar heating.

Secondary Electrons as an Energy Source for Life.

Life on Earth is found in a wide range of environments as long as the basic requirements of a liquid solvent, a nutrient source, and free energy are met. Previous hypotheses have speculated how extraterrestrial microbial life may function, among them that particle radiation might power living cells indirectly through radiolytic products. On Earth, so-called electrophilic organisms can harness electron flow from an extracellular cathode to build biomolecules. Here, we describe two hypothetical mechanisms, termed "direct electrophy" and "indirect electrophy" or "fluorosynthesis," by which organisms could harness extracellular free electrons to synthesize organic matter, thus expanding the ensemble of potential habitats in which extraterrestrial organisms might be found in the Solar System and beyond. The first mechanism involves the direct flow of secondary electrons from particle radiation to a microbial cell to power the organism. The second involves the indirect utilization of impinging secondary electrons and a fluorescing molecule, either biotic or abiotic in origin, to drive photosynthesis. Both mechanisms involve the attenuation of an incoming particle's energy to create low-energy secondary electrons. The validity of the hypotheses is assessed through simple calculations showing the biomass density attainable from the energy supplied. Also discussed are potential survival strategies that could be used by organisms living in possible habitats with a plentiful supply of secondary electrons, such as near the surface of an icy moon. While we acknowledge that the only definitive test for the hypothesis is to collect specimens, we also describe experiments or terrestrial observations that could support or nullify the hypotheses. Key Words: Radiation-Electrophiles-Subsurface life. Astrobiology 18, 73-85.

Force-induced transcellular tunnel formation in endothelial cells.

The endothelium serves as a protective semipermeable barrier in blood vessels and lymphatic vessels. Leukocytes and pathogens can pass directly through the endothelium by opening holes in endothelial cells, known as transcellular tunnels, which are formed by contact and self-fusion of the apical and basal plasma membranes. Here we test the hypothesis that the actin cytoskeleton is the primary barrier to transcellular tunnel formation using a combination of atomic force microscopy and fluorescence microscopy of live cells. We find that localized mechanical forces are sufficient to induce the formation of transcellular tunnels in HUVECs. When HUVECs are exposed to the bacterial toxin EDIN, which can induce spontaneous transcellular tunnels, less mechanical work is required to form tunnels due to the reduced cytoskeletal stiffness and thickness of these cells, similar to the effects of a ROCK inhibitor. We also observe actin enrichment in response to mechanical indentation that is reduced in cells exposed to the bacterial toxin. Our study shows that the actin cytoskeleton of endothelial cells provides both passive and active resistance against transcellular tunnel formation, serving as a mechanical barrier that can be overcome by mechanical force as well as disruption of the cytoskeleton.

Multicellular architecture of malignant breast epithelia influences mechanics.

Cell-matrix and cell-cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the "perceived stiffness" of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing.

Tensional homeostasis in single fibroblasts.

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.

Contractile equilibration of single cells to step changes in extracellular stiffness.

Extracellular stiffness has been shown to alter long timescale cell behaviors such as growth and differentiation, but the cellular response to changes in stiffness on short timescales is poorly understood. By studying the contractile response of cells to dynamic stiffness conditions using an atomic force microscope, we observe a seconds-timescale response to a step change in extracellular stiffness. Specifically, we observe acceleration in contraction velocity (µm/min) and force rate (nN/min) upon a step decrease in stiffness and deceleration upon a step increase in stiffness. Interestingly, this seconds-timescale response to a change in extracellular stiffness is not altered by inhibiting focal adhesion signaling or stretch-activated ion channels and is independent of cell height and contraction force. Rather, the response timescale is altered only by disrupting cytoskeletal mechanics and is well described by a simple mechanical model of a constant velocity actuator pulling against an internal cellular viscoelastic network. Consistent with the predictions of this model, we find that an osmotically expanding hydrogel responds to step changes in extracellular stiffness in a similar manner to cells. We therefore propose that an initial event in stiffness sensing is establishment of a mechanical equilibrium that balances contraction of the viscoelastic cytoskeleton with deformation of the extracellular matrix.

An AFM-based stiffness clamp for dynamic control of rigidity.

Atomic force microscopy (AFM) has become a powerful tool for measuring material properties in biology and imposing mechanical boundary conditions on samples from single molecules to cells and tissues. Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a 'force clamp' or 'position clamp'. However, stiffness, the third variable in the Hookean relation F = kx that describes AFM cantilever deflection, has not been dynamically controllable in the same way. Here we present and demonstrate a 'stiffness clamp' that can vary the apparent stiffness of an AFM cantilever. This method, employable on any AFM system by modifying feedback control of the cantilever, allows rapid and reversible tuning of the stiffness exposed to the sample in a way that can decouple the role of stiffness from force and deformation. We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel. We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1-100 nN/µm. This response was independent of the absolute contractile force and cell height, demonstrating that cells can react directly to changes in stiffness alone. In contrast, the hydrogel used in our experiment maintained a constant expansion velocity (dx/dt) over this range of stiffness, while the traction rate (dF/dt) changed with stiffness, showing that passive materials can also behave differently in different stiffness environments. The AFM stiffness clamp presented here, which is applicable to mechanical measurements on both biological and non-biological samples, may be used to investigate cellular mechanotransduction under a wide range of controlled mechanical boundary conditions.

Mechanics and contraction dynamics of single platelets and implications for clot stiffening.

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.