The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment.

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges' role in Giardia biology. Live imaging revealed that the flange grows to around 1 µm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia's unconventional actin cytoskeleton has an important role in supporting parasite attachment.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

De novo design of a fluorescence-activating ß-barrel.

The regular arrangements of ß-strands around a central axis in ß-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with ß-barrels. Here we show that accurate de novo design of ß-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ß-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.

World Trade Center dust induces nasal and neurological tissue injury while propagating reduced olfaction capabilities and increased anxiety behaviors.

Objective: Previous in vitro and in vivo World Trade Center particulate matter (WTCPM) exposure studies have provided evidence of exposure-driven oxidative/nitrative stress and inflammation on respiratory tract and aortic tissues. What remains to be fully understood are secondary organ impacts due to WTCPM exposure. This study was designed to test if WTC particle-induced nasal and neurologic tissue injury may result in unforeseen functional and behavioral outcomes.Material and Methods: WTCPM was intranasally administered in mice, evaluating genotypic, histopathologic, and olfaction latency endpoints.Results: WTCPM exposure was found to incite neurologic injury and olfaction latency in intranasally (IN) exposed mice. Single high-dose and repeat low-dose nasal cavity insults from WTCPM dust resulted in significant olfaction delays and enduring olfaction deficits. Anxiety-dependent behaviors also occurred in mice experiencing olfaction loss including significant body weight loss, increased incidence and time spent in hind stretch postures, as well as increased stationary time and decreased exploratory time. Additionally, WTCPM exposure resulted in increased whole brain wet/dry ratios and wet whole brain to body mass ratios that were correlated with exposure and increased exposure dose (p<0.05).Discussion: The potential molecular drivers of WTCPM-driven tissue injury and olfaction latency may be linked to oxidative/nitrative stress and inflammatory cascades in both upper respiratory nasal and brain tissues.Conclusion: Cumulatively, these data provide evidence of WTCPM exposure in relation to tissue damage related to oxidative stress-driven inflammation identified in the nasal cavity, propagated to olfactory bulb tissues and, potentially, over extended periods, to other CNS tissues.

Podocyte Aging: Why and How Getting Old Matters.

The effects of healthy aging on the kidney, and how these effects intersect with superimposed diseases, are highly relevant in the context of the population's increasing longevity. Age-associated changes to podocytes, which are terminally differentiated glomerular epithelial cells, adversely affect kidney health. This review discusses the molecular and cellular mechanisms underlying podocyte aging, how these mechanisms might be augmented by disease in the aged kidney, and approaches to mitigate progressive damage to podocytes. Furthermore, we address how biologic pathways such as those associated with cellular growth confound aging in humans and rodents.

Imaging nanobubble nucleation and hydrogen spillover during electrocatalytic water splitting.

Nucleation and growth of hydrogen nanobubbles are key initial steps in electrochemical water splitting. These processes remain largely unexplored due to a lack of proper tools to probe the nanobubble's interfacial structure with sufficient spatial and temporal resolution. We report the use of superresolution microscopy to image transient formation and growth of single hydrogen nanobubbles at the electrode/solution interface during electrocatalytic water splitting. We found hydrogen nanobubbles can be generated even at very early stages in water electrolysis, i.e., ∼500 mV before reaching its thermodynamic reduction potential. The ability to image single nanobubbles on an electrode enabled us to observe in real time the process of hydrogen spillover from ultrathin gold nanocatalysts supported on indium-tin oxide.

World Trade Center Dust induces airway inflammation while promoting aortic endothelial dysfunction.

Respiratory ailments have plagued occupational and public health communities exposed to World Trade Center (WTC) dust since the September 11, 2001 attack on the Twin Towers in Lower Manhattan. We proposed that these ailments were proposed to be induced by inhalation exposure to WTC particulate matter (WTCPM), that was released during the collapse of the buildings and its subsequent resuspension during cleanup. We investigated this hypothesis using both an in vitro and an in vivo mouse intranasal (IN) exposure models to identify the inflammatory potential of WTCPM with specific emphasis on respiratory and endothelial tissue responses. The in vitro exposure studies found WTCPM exposure to be positively correlated with cytotoxicity and increased NO2- production in both BEAS-2B pulmonary epithelial cells and THP-1 macrophage cells. The in vivo C57BL/6 mouse studies found significant increases in inflammatory markers including increases in polymorphonuclear neutrophil (PMN) influx into nasal and bronchoalveolar lavage fluids (NLF and BALF), as well as increased levels of total protein and cytokine/chemokines levels. Concurrently, NLF, BALF, and serum NO2- levels exhibited significant homeostatic temporal deviations as well as temporal myograohic aortic dysfunction in myography studies. Respiratory exposure to- and evidence -based retention of- WTCPM may have contributed to chronic systemic effects in exposed mice that r resembled to observed effects in WTCPM-exposed human populations. Collectively, these findings are reflective of WTCPM exposure and its effect(s) on respiratory and aortic tissues, highlighting potential dysfunctional pathways that may precipitate inflammatory events, while simultaneously altering homeostatic balances. The tight interplay between these balances, when chronically altered, may contribute to- or result in- chronically diseased pathological states.

Switchable Fluorophores for Single-Molecule Localization Microscopy.

The past decade has witnessed an explosion in the use of super-resolution fluorescence microscopy methods in biology and other fields. Single-molecule localization microscopy (SMLM) is one of the most widespread of these methods and owes its success in large part to the ability to control the on-off state of fluorophores through various chemical, photochemical, or binding-unbinding mechanisms. We provide here a comprehensive overview of switchable fluorophores in SMLM including a detailed review of all major classes of SMLM fluorophores, and we also address strategies for labeling specimens, considerations for multichannel and live-cell imaging, potential pitfalls, and areas for future development.

Dual lineage tracing shows that glomerular parietal epithelial cells can transdifferentiate toward the adult podocyte fate.

Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman's capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.

Expansion microscopy with conventional antibodies and fluorescent proteins.

Expansion microscopy is a technique in which fluorophores on fixed specimens are linked to a swellable polymer that is physically expanded to enable super-resolution microscopy with ordinary microscopes. We have developed and characterized new methods for linking fluorophores to the polymer that now enable expansion microscopy with conventional fluorescently labeled antibodies and fluorescent proteins. Our methods simplify the procedure and expand the palette of compatible labels, allowing rapid dissemination of the technique.

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM.

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.

Single-Molecule Electrochemistry on a Porous Silica-Coated Electrode.

Here we report the direct observation and quantitative analysis of single redox events on a modified indium-tin oxide (ITO) electrode. The key in the observation of single redox events are the use of a fluorogenic redox species and the nanoconfinement and hindered redox diffusion inside 3-nm-diameter silica nanochannels. A simple electrochemical process was used to grow an ultrathin silica film (∼100 nm) consisting of highly ordered parallel nanochannels exposing the electrode surface from the bottom. The electrode-supported 3-nm-diameter nanochannels temporally trap fluorescent resorufin molecules resulting in hindered molecular diffusion in the vicinity of the electrode surface. Adsorption, desorption, and heterogeneous redox events of individual resorufin molecules can be studied using total-internal reflection fluorescence (TIRF). The rate constants of adsorption and desorption processes of resorufin were characterized from single-molecule analysis to be (1.73 ± 0.08) × 10-4 cm·s-1 and 15.71 ± 0.76 s-1, respectively. The redox events of resorufin to the non-fluorescent dihydroresorufin were investigated by analyzing the change in surface population of single resorufin molecules with applied potential. The scan-rate-dependent molecular counting results (single-molecule fluorescence voltammetry) indicated a surface-controlled electrochemical kinetics of the resorufin reduction on the modified ITO electrode. This study demonstrates the great potential of mesoporous silica as a useful modification scheme for studying single redox events on a variety of transparent substrates such as ITO electrodes and gold or carbon film coated glass electrodes. The ability to electrochemically grow and transfer mesoporous silica films onto other substrates makes them an attractive material for future studies in spatial heterogeneity of electrocatalytic surfaces.

A model for the generation and interconversion of ER morphologies.

The peripheral endoplasmic reticulum (ER) forms different morphologies composed of tubules and sheets. Proteins such as the reticulons shape the ER by stabilizing the high membrane curvature in cross-sections of tubules and sheet edges. Here, we show that membrane curvature along the edge lines is also critical for ER shaping. We describe a theoretical model that explains virtually all observed ER morphologies. The model is based on two types of curvature-stabilizing proteins that generate either straight or negatively curved edge lines (R- and S-type proteins). Dependent on the concentrations of R- and S-type proteins, membrane morphologies can be generated that consist of tubules, sheets, sheet fenestrations, and sheet stacks with helicoidal connections. We propose that reticulons 4a/b are representatives of R-type proteins that favor tubules and outer edges of sheets. Lunapark is an example of S-type proteins that promote junctions between tubules and sheets. In a tubular ER network, lunapark stabilizes three-way junctions, i.e., small triangular sheets with concave edges. The model agrees with experimental observations and explains how curvature-stabilizing proteins determine ER morphology.

Ultrabright photoactivatable fluorophores created by reductive caging.

Sub-diffraction-limit imaging can be achieved by sequential localization of photoactivatable fluorophores, for which the image resolution depends on the number of photons detected per localization. We report a strategy for fluorophore caging that creates photoactivatable probes with high photon yields. Upon photoactivation, these probes can provide 10(4)-10(6) photons per localization and allow imaging of fixed samples with resolutions of several nanometers. This strategy can be applied to many fluorophores across the visible spectrum.

Tungsten-induced carcinogenesis in human bronchial epithelial cells.

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans.

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats.

Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12-13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6-11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

An end-to-end workflow for nondestructive 3D pathology.

Recent advances in 3D pathology offer the ability to image orders of magnitude more tissue than conventional pathology methods while also providing a volumetric context that is not achievable with 2D tissue sections, and all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis, however, is not trivial and requires careful attention to a series of details during tissue preparation, imaging and initial data processing, as well as iterative optimization of the entire process. Here, we provide an end-to-end procedure covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. Although 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol focuses on the use of a fluorescent analog of hematoxylin and eosin, which remains the most common stain used for gold-standard pathological reports. We present our guidelines for a broad range of end users (e.g., biologists, clinical researchers and engineers) in a simple format. The end-to-end workflow requires 3-6 d to complete, bearing in mind that data analysis may take longer.