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We encounter titanium dioxide nanoparticles (TiO 2 NPs) throughout our daily lives in the form of food coloring, cosmetics, and industrial materials. They are used on a massive industrial scale, with over 1 million metric tons in the global market. For the workers who process these materials, inhalation is a major concern. The goal of our current research is to provide a direct comparison of the three major types of TiO 2 NPs (P25, E171, R101) in terms of surface characterization, cellular response, and in vivo response following introduction into the lungs of mice. In both cellular and in vivo experiments, we observe a pro-inflammatory response to the P25 TiO 2 NPs that is not observed in the E171 or R101 TiO 2 NPs at mass-matched concentrations. Cellular experiments measured a cytokine, TNF-α, as a marker of a pro-inflammatory response. In vivo experiments in mice measured the number of immune cells and four pro-inflammatory cytokines (IL-6, MIP-2, IP-10, and MCP-1) present in bronchoalveolar lavage fluid. A detailed physical and chemical characterization of the TiO 2 NPs shows that the P25 TiO 2 NPs are distinguished by smaller primary particles suggesting that samples matched by mass contain a larger number of P25 TiO 2 NPs. Cellular dose-response measurements with the P25, E171, and R101 TiO 2 NPs support this hypothesis showing increased TNF-α release by macrophages as a function of TiO 2 NP dose. Overall, this direct comparison of the three major types of TiO 2 NPs shows that the number of particles in a dose, which is dependent on the particle diameter, is a key parameter in TiO 2 NP-induced inflammation.

The increasing use of multiwalled carbon nanotubes (MWCNTs) could increase the risk of allergic lung disease in occupational or consumer settings. We previously reported that MWCNTs exacerbated allergic lung disease in mice induced by extract from house dust mites (HDM), a common cause of asthma in humans. Because MWCNTs avidly bind biomolecules to form protein coronas that can modify immunotoxicity, we hypothesized that exacerbation of allergic lung disease in mice caused by coexposure to MWCNTs and HDM extract was due to the formation of an allergen corona. In a first set of experiments, male and female C57BL/6J mice were coexposed to MWCNTs and HDM extract over 3 weeks compared to MWCNTs or HDM extract alone. In a second set of experiments, mice were exposed to pristine MWCNTs or MWCNTs with an HDM allergen corona (HDM-MWCNTs). HDM-MWCNTs were formed by incubating MWCNTs with HDM extract, where ∼7% of proteins adsorbed to MWCNTs, including Der p 1 and Der p 2. At necropsy, bronchoalveolar lavage fluid was collected from lungs to assess lactate dehydrogenase, total protein and inflammatory cells, while lung tissue was used for histopathology, qPCR, and Western blotting. Compared to MWCNTs or HDM extract alone, coexposure to MWCNTs and HDM extract or exposure to HDM-MWCNTs increased pathological outcomes associated with allergic lung disease (eosinophilia, fibrosis, mucous cell metaplasia), increased mRNAs associated with fibrosis ( Col1A1 , Arg1 ) and enhanced STAT6 phosphorylation in lung tissue. These findings indicated that exacerbation of HDM-induced allergic lung disease by MWCNTs is due to an allergen corona.

Autoimmune and inflammatory diseases are highly complex, limiting treatment and the development of new therapies. Recent work has shown that cell-free DNA bound to biological microparticles is linked to systemic lupus erythematosus, a prototypic autoimmune disease. However, the heterogeneity and technical challenges associated with the study of biological particles have hindered a mechanistic understanding of their role. Our goal was to develop a well-controlled DNA-particle model system to understand how DNA-particle complexes affect cells. We first characterized the adsorption of DNA on the surface of polystyrene nanoparticles (200 nm and 2 µm) using transmission electron microscopy, dynamic light scattering, and colorimetric DNA concentration assays. We found that DNA adsorbed on the surface of nanoparticles was resistant to degradation by DNase 1. Macrophage cells incubated with the DNA-nanoparticle complexes had increased production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We probed two intracellular DNA sensing pathways, toll-like receptor 9 (TLR9) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), to determine how cells sense the DNA-nanoparticle complexes. We found that the cGAS-STING pathway is the primary route for the interaction between DNA-nanoparticles and macrophages. These studies provide a molecular and cellular-level understanding of DNA-nanoparticle-macrophage interactions. In addition, this work provides the mechanistic information necessary for future in vivo experiments to elucidate the role of DNA-particle interactions in autoimmune diseases, providing a unique experimental framework to develop novel therapeutic approaches., Competing Interests: Competing interests statement:The authors declare no competing interest.

Multi-walled carbons nanotubes (MWCNTs) are used in materials for the construction, automotive, and aerospace industries. Workers and consumers are exposed to these materials via inhalation. Existing recommended exposure limits are based on MWCNT exposures that do not take into account more realistic co-exposures. Our goal was to understand how a common allergen, house dust mites, interacts with pristine MWCNTs and lung fluid proteins. We used gel electrophoresis, western blotting, and proteomics to characterize the composition of the allergen corona formed from house dust mite extract on the surface of MWCNTs. We found that the corona is dominated by der p 2, a protein associated with human allergic responses to house dust mites. Der p 2 remains adsorbed on the surface of the MWCNTs following subsequent exposures to lung fluid proteins. The high concentration of der p 2, localized on surface of MWCNTs, has important implications for house dust mite-induced allergies and asthma. This research provides a detailed characterization of the complex house dust mite-lung fluid protein coronas for future cellular and in vivo studies. These studies will help to address the molecular and biochemical mechanisms underlying the exacerbation of allergic lung disease by nanomaterials., Competing Interests: Ethics Declarations Conflicts of interest The authors declare that they have no conflicts of interest.

Inhalation is a major exposure route to nanoparticles. Following inhalation, nanoparticles first interact with the lung lining fluid, a complex mixture of proteins, lipids, and mucins. We measure the concentration and composition of lung fluid proteins adsorbed on the surface of titanium dioxide (TiO 2 ) nanoparticles. Using proteomics, we find that lung fluid results in a unique protein corona on the surface of the TiO 2 nanoparticles. We then measure the expression of three cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)) associated with lung inflammation. We find that the corona formed from lung fluid leads to elevated expression of these cytokines in comparison to bare TiO 2 nanoparticles or coronas formed from serum or albumin. These experiments show that understanding the concentration and composition of the protein corona is essential for understanding the pulmonary response associated with human exposure to nanoparticles.

Essential cellular processes such as metabolism, protein synthesis, and autophagy require the intracellular transport of membrane-bound vesicles. The importance of the cytoskeleton and associated molecular motors for transport is well documented. Recent research has suggested that the endoplasmic reticulum (ER) may also play a role in vesicle transport through a tethering of vesicles to the ER. We use single-particle tracking fluorescence microscopy and a Bayesian change-point algorithm to characterize vesicle motility in response to the disruption of the ER, actin, and microtubules. This high-throughput change-point algorithm allows us to efficiently analyze thousands of trajectory segments. We find that palmitate-mediated disruption of the ER leads to a significant decrease in vesicle motility. A comparison with the disruption of actin and microtubules shows that disruption of the ER has a significant impact on vesicle motility, greater than the disruption of actin. Vesicle motility was dependent on cellular region, with greater motility in the cell periphery than the perinuclear region, possibly due to regional differences in actin and the ER. Overall, these results suggest that the ER is an important factor in vesicle transport., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Nanoparticles in contact with proteins form a "corona" of proteins adsorbed on the nanoparticle surface. Subsequent biological responses are then mediated by the adsorbed proteins rather than the bare nanoparticles. The use of nanoparticles as nanomedicines and biosensors would be greatly improved if researchers were able to predict which specific proteins will adsorb on a nanoparticle surface. We use a recently developed automated workflow with a liquid handling robot and low-cost proteomics to determine the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. In addition to these findings, we describe two methodological advances to help reduce the cost associated with protein corona experiments. We have automated the digest step necessary for proteomics and measured the variability between triplicate samples at each stage of the proteomics experiments. Overall, these results demonstrate the importance of understanding the multiple parameters that influence corona formation, provide new tools for corona characterization, and advance bioanalytical research in nanomaterials., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Lysosomes are membrane-bound organelles that serve as the endpoint for endocytosis, phagocytosis, and autophagy, degrading the molecules, pathogens, and organelles localized within them. These cellular functions require intracellular transport. We use fluorescence microscopy to characterize the motion of lysosomes as a function of intracellular region, perinuclear or periphery, and lysosome diameter. Single-particle tracking data are complemented by changepoint identification and analysis of a mathematical model for state switching. We first classify lysosomal motion as motile or stationary. We then study how lysosome location and diameter affects the proportion of time spent in each state and quantify the speed during motile periods. We find that the proportion of time spent stationary is strongly region dependent, with significantly decreased motility in the perinuclear region. Increased lysosome diameter only slightly decreases speed. Overall, these results demonstrate the importance of decomposing particle trajectories into qualitatively different behaviors before conducting population-wide statistical analysis. Our results suggest that intracellular region is an important factor to consider in studies of intracellular transport., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: The growing field of bacterial electrophysiology examines the relationship between bacterial membrane potential and cell division, growth, sporulation, and biofilm formation. These experiments require Nernstian fluorescent dyes to monitor membrane potential. Our research uses single cell imaging to determine if a common fluorescent dye, Thioflavin T (ThT), affects the growth of bacteria., Materials and Methods: We use a combination of standard growth curve measurements and single cell imaging, both brightfield and fluorescence microscopy, to monitor the growth of Bacillus subtilis and Escherichia coli as a function of ThT concentration. Increased membrane potential (hyperpolarization) leads to increased intracellular accumulation of ThT: High fluorescence intensity is an indicator of hyperpolarization. Blue light is used to hyperpolarize a subpopulation of cells to monitor cellular elongation in response to increased cellular internalization of ThT., Results: Single cell imaging shows that the elongation rates of B. subtilis and E. coli are decreased when these cells are incubated with ThT. At micromolar concentrations of ThT, this effect may be masked in standard growth curves, but is visible with single cell measurements on agarose pads., Conclusions: The increased cellular accumulation of ThT is a standard measure of hyperpolarization in bacterial electrophysiology. Growth curves, a bulk measurement, are typically used to determine suitable concentrations of ThT for use in experiments. Single cell measurements show that cells incubated with ThT have decreased elongation rates. This creates a potential experimental artifact that could lead to misinterpretation of data. Hyperpolarized cells internalize more ThT. This increased intracellular concentration of ThT, rather than the change in membrane potential, could lead to decreased growth. These experiments point toward the importance of single cell measurements to detect subtle changes in cell growth. We hope this research will be useful for other researchers in their choice of dye for the detection of membrane potential., (Copyright 2022, Mary Ann Liebert, Inc., publishers.)

Titanium dioxide (TiO 2 ) particles are a common ingredient in food, providing the bright white color for many candies, gums, and frostings. While ingestion of these materials has been examined previously, few studies have examined the effect of these particles on lung cells. Inhalation is an important exposure pathway for workers processing these foods and, more recently, home users who purchase these particles directly. We examine the response of lung cells to food-grade TiO 2 particles using a combination of fluorescence microscopy and RT-PCR. These experiments show that TiO 2 particles generate intracellular reactive oxygen species, specifically superoxide, and alter expression of two epigenetic modifiers, histone deacetylase 9 (HDAC9) and HDAC10. We use a protein corona formed from superoxide dismutase (SOD), an enzyme that scavenges superoxide, to probe the relationship between TiO 2 particles and superoxide generation. These experiments show that low, non-cytotoxic, concentrations of food-grade TiO 2 particles lead to cellular responses, including altering two enzymes responsible for epigenetic modifications. This production of superoxide and change in epigenetic modifiers could affect human health following inhalation. We expect this research will motivate future in vivo experiments examining the pulmonary response to food-grade TiO 2 particles.

Introduction and Hypothesis: Obstetric fistulas have devastating consequences for women. Although surgical repair is largely successful in closing the defect, many women with successful fistula closure report persistent urinary incontinence. Our study is aimed at characterizing incontinence after successful fistula repair and its impact on quality of life., Methods: This cross-sectional study enrolled women with a history of successful obstetric fistula closure with (n = 51; cases) or without (n = 50; controls) persistent urinary incontinence. Data were collected in Mekelle, Ethiopia, between 2016 and 2018. All cases underwent clinical evaluation and completed questionnaires characterizing the type, severity, and impact of incontinence., Results: Cases were significantly more likely to have acquired their fistula at an earlier age and with their first vaginal delivery compared with controls. Almost all cases reported both stress (98%) and urgency (94%) incontinence, and half reported constant urinary leakage (49%) despite successful fistula closure. Of cases who completed urodynamic evaluation (n = 22), all had genuine stress incontinence and none had detrusor overactivity. All cases reported moderate to severe (80.4%) or very severe (19.6%) incontinence (measured by ICIQ-SF) and this had a moderate to severe negative impact on their quality of life (as measured by ICIQ-QoL). Although history of suicidal ideation was not significantly different between the groups, among those with suicidal ideation, cases were more likely to report having made a plan and/or attempted to commit suicide., Conclusions: When urinary incontinence persists after successful fistula closure, it tends to be severe and of mixed etiology and has a significant negative impact on quality of life and mental health.

There has been much recent interest in the protein "corona," the nonspecific adsorption of proteins on the surface of nanoparticles used in biological applications. This research investigates an analogous DNA corona. We find that particles (200 nm and 1 μm) incubated with DNA form a DNA corona, with a higher concentration of DNA adsorbed on the surface of cationic nanoparticles. With protein present, a combined DNA and protein corona is formed although DNA in solution displaces protein from the nanoparticle surface. Displacement of protein from the nanoparticle surface is dependent on the concentration of DNA in solution and was also observed for planar surfaces. Overall, we expect this investigation of the DNA corona to be important for nanomedicine applications, as well as disease states, especially systemic lupus erythematosus, in which biological particles with bound DNA are important mediators of inflammation and thrombosis.

Background: The use of electricity to mediate bacterial growth is unique in providing spatial control, but requires a more detailed understanding. Methods: We use two gold wires on a glass coverslip with an overlayer of agar to image Escherichia coli cells with brightfield and fluorescence microscopy while simultaneously applying a voltage. Cells outside of the wires provide a control population to measure cell growth as a function of voltage, rather than any difference in culture conditions or growth phase. Results: An applied voltage suppresses the fraction of E. coli undergoing elongation and division with recovery to control values when the voltage is removed. Depolarization is observed over the same voltage range suggesting a membrane potential-mediated response. Conclusions: Our experiments identify and use subcytotoxic voltages to measure differences in the fraction of E. coli cells elongating and dividing as a function of applied voltage. It is hoped that this research will inform the developing field of bacterial electrophysiology., Competing Interests: No competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

Nanoparticles used in biological settings are exposed to proteins that adsorb on the surface forming a protein corona. These adsorbed proteins dictate the subsequent cellular response. A major challenge has been predicting what proteins will adsorb on a given nanoparticle surface. Instead, each new nanoparticle and nanoparticle modification must be tested experimentally to determine what proteins adsorb on the surface. We propose that any future predictive ability will depend on large datasets of protein-nanoparticle interactions. As a first step towards this goal, we have developed an automated workflow using a liquid handling robot to form and isolate protein coronas. As this workflow depends on magnetic separation steps, we test the ability to embed magnetic nanoparticles within a protein nanoparticle. These experiments demonstrate that magnetic separation could be used for any type of nanoparticle in which a magnetic core can be embedded. Higher-throughput corona characterization will also require lower-cost approaches to proteomics. We report a comparison of fast, low-cost, and standard, slower, higher-cost liquid chromatography coupled with mass spectrometry to identify the protein corona. These methods will provide a step forward in the acquisition of the large datasets necessary to predict nanoparticle-protein interactions.

Titanium dioxide (TiO 2 ) nanoparticles are used on a massive scale in commercial and industrial products. Of specific concern is how the inhalation of these nanoparticles in a manufacturing setting may affect human health. We examine the cellular response to TiO 2 nanoparticles using a combination of cell-free spectroscopic assays, fluorescence microscopy, Western blotting, and TiO 2 nanoparticle surface modifications. These experiments show that TiO 2 nanoparticles generate superoxide, both in solution and in cells, and this intracellular superoxide decreases expression of histone deacetylase 9 (HDAC9), an epigenetic modifier. We use protein coronas formed from superoxide dismutase (SOD) and catalase, enzymes that scavenge reactive oxygen species (ROS), to probe the relationship between TiO 2 nanoparticles, ROS, and the subsequent cellular response. These protein coronas provide nanoparticle-localized scavengers that demonstrate that the nanoparticles are the source of the intracellular superoxide. Importantly, the use of a SOD corona or surface passivated TiO 2 nanoparticles prevents the decrease of HDAC9. These experiments elucidate the underlying mechanism of TiO 2 nanoparticle-mediated cellular responses including oxidative stress and changes in gene expression. They also provide the first demonstration of a protein corona as a tool for probing cellular responses to nanoparticles. Overall, this research shows that low, nontoxic concentrations of TiO 2 nanoparticles alter an enzyme responsible for epigenetic modifications, which points to concerns regarding long-term exposures in manufacturing settings.

Objective: Obstetric fistula is a devastating childbirth injury that leaves women incontinent, stigmatized and often isolated from their families and communities. In Ethiopia, although much attention has focused on treating and preventing obstetric fistula, other more prevalent childbirth-related pelvic floor disorders, such as pelvic organ prolapse, non-fistula-related incontinence and post-fistula residual incontinence, remain largely unattended. The lack of international and local attention to addressing devastating pelvic floor disorders is concerning for women in low- and middle-income countries. The objective of this article is to highlight the need for a more comprehsive approach to pelvic floor care and to share our experience in addressing it., Methods: Here, we share our experience launching one of the first formal training programs in Female Pelvic Medicine and Reconstructive Surgery (FPMRS) in Ethiopia., Results: This fellowship program provides quality care while strengthening the health system in its local context. This program has positioned Ethiopia to be a regional leader by providing comprehensive training of surgeons and allied health professionals, building appropriate health system and research infrastructure, and developing a formal FPMRS training curriculum., Conclusion: We hope that sharing this experience will serve as a template for others championing comprehensive pelvic floor care for women in low- and middle-income countries.

Nanoparticles present in any biological environment are exposed to extracellular proteins. These proteins adsorb on the surface of the nanoparticle forming a "protein corona." These proteins control the interaction of nanoparticles with cells. The interaction of proteins with the nanoparticle surface is governed by physical chemistry. Understanding this process requires spectroscopy, microscopy, and computational tools that are familiar to physical chemists. This perspective provides an overview of the protein corona along with two future directions: first, the need for new computational approaches, including machine learning, to predict corona formation and second, the extension of protein corona studies to more complex environments ranging from lung fluids to waste water treatment.

Understanding the cellular basis of human health and disease requires the spatial resolution of microscopy and the molecular-level details provided by spectroscopy. This review highlights imaging methods at the intersection of microscopy and spectroscopy with applications in cell biology. Imaging methods are divided into three broad categories: fluorescence microscopy, label-free approaches, and imaging tools that can be applied to multiple imaging modalities. Just as these imaging methods allow researchers to address new biological questions, progress in biological sciences will drive the development of new imaging methods. We highlight four topics in cell biology that illustrate the need for new imaging tools: nanoparticle-cell interactions, intracellular redox chemistry, neuroscience, and the increasing use of spheroids and organoids. Overall, our goal is to provide a brief overview of individual imaging methods and highlight recent advances in the use of microscopy for cell biology.

The goal of this review is to highlight materials that show exciting promise for either entirely new cellular-level applications or new approaches to long-standing biological challenges. The authors start with two more established materials, graphene and carbon nanotubes, and then progress to conducting polymers, followed by an overview of the microresonators, nanowires, and spasers used as intracellular lasers. These materials provide new approaches to gene and drug delivery, cellular regeneration, mechanical sensing, imaging, and the modulation and recording of cellular activity. Of specific interest is the comparison of these materials with existing technologies, the method of cellular delivery, and the all-encompassing challenge of biocompatibility. Concluding remarks examine the extension of these materials from cellular-level experiments to in vivo applications, including the method of activation: light, electricity, and ultrasound. Overall, these materials and their associated applications illustrate the most recent advances in material-cell interactions.

Female genital mutilation/cutting (FGM/C)-also known as Female Genital Cutting or Mutilation-is defined as the partial or total removal of the female external genitalia for non-therapeutic reasons. This White Paper, prepared under the auspices of the International Continence Society (ICS), is intended by the ICS as a statement promoting the abandonment of this practice. The ICS also supports the respectful and evidence-based care or treatment of women and girls already affected by FGM/C, in keeping with the World Health Organization (WHO) Guidelines on the Management of Health Complications from Female Genital Mutilation. Our members specialize in pelvic floor disorders from perspectives within a range of specialties; we encounter and treat women living with FGM/C and its consequences-particularly incontinence, infections, voiding dysfunction, sexual dysfunction, chronic pelvic pain, and obstetric trauma. Understanding the ethical, sociocultural, medical and surgical factors surrounding FGM/C is central to caring for women and girls with a history of FGM/C. The ICS voices herein state strong opposition to FGM/C. We encourage members to apply their skills to improve prevention strategies and the management of those affected., (© 2019 Wiley Periodicals, Inc.)

TiO 2 nanoparticles are widely used in consumer products and industrial applications, yet little is understood regarding how the inhalation of these nanoparticles impacts long-term health. This is especially important for the occupational safety of workers who process these materials. We used RNA sequencing to probe changes in gene expression and fluorescence microscopy to image intracellular reactive oxygen species (ROS) in human lung cells incubated with low, non-cytotoxic, concentrations of TiO 2 nanoparticles. Experiments were designed to measure changes in gene expression following an acute exposure to TiO 2 nanoparticles and changes inherited by progeny cells. We observe that TiO 2 nanoparticles lead to significant (>2000 differentially expressed genes) changes in gene expression following a 24 hour incubation. Following this acute exposure, the response dissipates with only 34 differentially expressed genes in progeny cells. The progeny cells adapt to this initial exposure, observed when re-challenged with a second acute TiO 2 nanoparticle exposure. Accompanying these changes in gene expression is the production of intracellular ROS, specifically superoxide, along with changes in oxidative stress-related genes. These experiments suggest that TiO 2 nanoparticles adapt to oxidative stress through transcriptional changes over multiple generations of cells., Competing Interests: Conflicts of interest The authors declare no conflict of interest.

Nanoparticles used in cellular applications encounter free serum proteins that adsorb onto the surface of the nanoparticle, forming a protein corona. This protein layer controls the interaction of nanoparticles with cells. For nanomedicine applications, it is important to consider how intravenous injection and the subsequent shear flow will affect the protein corona. Our goal was to determine if shear flow changed the composition of the protein corona and if these changes affected cellular binding. Colorimetric assays of protein concentration and gel electrophoresis demonstrate that polystyrene nanoparticles subjected to flow have a greater concentration of serum proteins adsorbed on the surface, especially plasminogen. Plasminogen, in the absence of nanoparticles, undergoes changes in structure in response to flow, characterized by fluorescence and circular dichroism spectroscopy. The protein-nanoparticle complexes formed from fetal bovine serum after flow had decreased cellular binding, as measured with flow cytometry. In addition to the relevance for nanomedicine, these results also highlight the technical challenges of protein corona studies. The composition of the protein corona was highly dependent on the initial mixing step: rocking, vortexing, or flow. Overall, these results reaffirm the importance of the protein corona in nanoparticle-cell interactions and point toward the challenges of predicting corona composition based on nanoparticle properties., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Introduction: The terminology for adult neurogenic lower urinary tract dysfunction (ANLUTD) should be defined and organized in a clinically based consensus Report., Methods: This Report has been created by a Working Group under the auspices and guidelines of the International Continence Society (ICS) Standardization Steering Committee (SSC) assisted at intervals by external referees. All relevant definitions for ANLUTD were updated on the basis of research over the last 14 years. An extensive process of 18 rounds of internal and external review was involved to exhaustively examine each definition, with decision-making by collective opinion (consensus)., Results: A Terminology Report for ANLUTD, encompassing 97 definitions (42 NEW and 8 CHANGED, has been developed. It is clinically based with the most common diagnoses defined. Clarity and user-friendliness have been key aims to make it interpretable by practitioners and trainees in all the different groups involved not only in lower urinary tract dysfunction but additionally in many other medical specialties., Conclusion: A consensus-based Terminology Report for ANLUTD has been produced to aid clinical practice and research., (© 2017 Wiley Periodicals, Inc.)

Nanoparticles, especially metal oxide nanoparticles, are used in a wide range of commercial and industrial applications that result in direct human contact, such as titanium dioxide nanoparticles in paints, food colorings, and cosmetics, or indirectly through release of nanoparticle-containing materials into the environment. Workers who process nanoparticles for downstream applications are exposed to especially high concentrations of nanoparticles. For physical chemists, nanoparticles present an interesting area of study as the small size of nanoparticles changes the properties from that of the bulk material, leading to novel properties and reactivity. For the public health community, this reduction in particle size means that exposure limits and outcomes that were determined from bulk material properties are not necessarily valid. Informed determination of exposure limits requires a fundamental understanding of how nanoparticles interact with cells. This Feature Article highlights the areas of intersection between physical chemistry and public health in understanding nanoparticle-cell interactions, with a focus on titanium dioxide nanoparticles. It provides an overview of recent research examining the interaction of titanium dioxide nanoparticles with cells in the absence of UV light and provides recommendations for additional nanoparticle-cell research in which physical chemistry expertise could help to inform the public health community.

The last 2 decades have brought an explosion of research, new products, and general attention to the problem of urinary urgency, and yet patients continue to be plagued by this symptom - especially the elderly. What is it? What does it mean? How can we guide patients successfully through this challenge? This paper presents a review of current thinking about urgency relevant to the practicing clinician, including the epidemiology, pathophysiology, evaluation, and treatment of these patients., (© 2018 S. Karger AG, Basel.)

Titanium dioxide (TiO 2 ) nanoparticles, used as pigments and photocatalysts, are widely present in modern society. Inhalation or ingestion of these nanoparticles can lead to cellular-level interactions. We examined the very first step in this cellular interaction, the effect of TiO 2 nanoparticles on the lipids of the plasma membrane. Within 12 h of TiO 2 nanoparticle exposure, the lipids of the plasma membrane were oxidized, determined with a malondialdehyde assay. Lipid peroxidation was inhibited by surface passivation of the TiO 2 nanoparticles, incubation with an antioxidant (Trolox), and the presence of serum proteins in solution. Subsequent experiments determined that serum proteins adsorbed on the surface of the TiO 2 nanoparticles, forming a protein corona, inhibit lipid peroxidation. Super-resolution fluorescence microscopy showed that these serum proteins were clustered on the nanoparticle surface. These protein clusters slow lipid peroxidation, but by 24 h, the level of lipid peroxidation is similar, independent of the protein corona or free serum proteins. Additionally, over 24 h, this corona of proteins was displaced from the nanoparticle surface by free proteins in solution. Overall, these experiments provide the first mechanistic investigation of plasma membrane oxidation by TiO 2 nanoparticles, in the absence of UV light and as a function of the protein corona, approximating a physiological environment.

We describe the use of PEDOT:PSS conducting polymer microwires to modulate action potentials in single cells., Pedot: PSS conducting polymer microwires are electrochemically synthesized with diameters ranging from 860 nm to 4.5 μm and conductivities of ~30 S/cm. The length of the microwires is controlled by the spacing of the electrodes used for the electrochemical polymerization. We demonstrate the use of these microwires to control the action potentials of cardiomyocytes, showing that the cellular contractions match the frequency of the applied voltage. Membrane integrity assays confirm that the voltage delivered by the wires does not damage cells. We expect the conducting polymer microwires will be useful as minimally invasive devices to control the electrical properties of cells with high spatial precision.

All cells have a resting membrane potential resulting from an ion gradient across the plasma membrane. The resting membrane potential of cells is tightly coupled to regeneration and differentiation. The ability to control this parameter provides the opportunity for both biomedical advances and the probing of fundamental bioelectric pathways. The use of poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) conducting polymer microwires to depolarize cells is tested using E. coli cells loaded with a fluorescent dye that is pumped out of the cells in response to depolarization; a more positive membrane potential. Fluorescence imaging of the cells in response to a conducting-polymer-microwire applied voltage confirms depolarization and shows that the rate of depolarization is a function of the applied voltage and frequency. Microwire activity does not damage the cells, demonstrated with a propidium iodide assay of membrane integrity. The conducting polymer microwires do not penetrate the cell, or even come into contact with the cell; they only need to generate a minimum electric field, controlled by the placement of the wires. It is expected that these microwires will provide a new, noninvasive, cellular-scale tool for the control of resting membrane potential with high spatial precision., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Titanium dioxide nanoparticles (TiO 2 NPs), used as pigments and photocatalysts, are ubiquitous in our daily lives. Previous work has observed cellular oxidative stress in response to the UV-excitation of photocatalytic TiO 2 NPs. In comparison, most human exposure to TiO 2 NPs takes place in the dark, in the lung following inhalation or in the gut following consumption of TiO 2 NP food pigment. Our spectroscopic characterization shows that both photocatalytic and food grade TiO 2 NPs, in the dark, generate low levels of reactive oxygen species (ROS), specifically hydroxyl radicals and superoxides. These ROS oxidize serum proteins that form a corona of proteins on the NP surface. This protein layer is the interface between the NP and the cell. An oxidized protein corona triggers an oxidative stress response, detected with PCR and western blotting. Surface modification of TiO 2 NPs to increase or decrease surface defects correlates with ROS generation and oxidative stress, suggesting that NP surface defects, likely oxygen vacancies, are the underlying cause of TiO 2 NP-induced oxidative stress.

Interfacing devices with cells and tissues requires new nanoscale tools that are both flexible and electrically active. We demonstrate the use of PEDOT:PSS conducting polymer nanowires for the local control of protein concentration in water and biological media. We use fluorescence microscopy to compare the localization of serum albumin in response to electric fields generated by narrow (760 nm) and wide (1.5 μm) nanowires. We show that proteins in deionized water can be manipulated over a surprisingly large micron length scale and that this distance is a function of nanowire diameter. In addition, white noise can be introduced during the electrochemical synthesis of the nanowire to induce branches into the nanowire allowing a single device to control multiple nanowires. An analysis of growth speed and current density suggests that branching is due to the Mullins-Sekerka instability, ultimately controlled by the roughness of the nanowire surface. These small, flexible, conductive, and biologically compatible PEDOT:PSS nanowires provide a new tool for the electrical control of biological systems.

Cathepsins are powerful proteases, once referred to as the lysosomal cysteine proteases, that have been implicated in breast cancer invasion and metastasis, but pharmaceutical inhibitors have suffered failures in clinical trials due to adverse side effects. Scientific advancement from lysosomotropic to cell impermeable cathepsin inhibitors have improved efficacy in treating disease, but off-target effects have still been problematic, motivating a need to better understand cellular feedback and responses to treatment with cathepsin inhibitors. To address this need, we investigated effects of E-64 and cystatin C, two broad spectrum cathepsin inhibitors, on cathepsin levels intra- and extracellularly in MDA-MB-231 breast cancer cells. Cathepsins S and L had opposing responses to both E-64 and cystatin C inhibitor treatments with paradoxically elevated amounts of active cathepsin S, but decreased amounts of active cathepsin L, as determined by multiplex cathepsin zymography. This indicated cellular feedback to selectively sustain the amounts of active cathepsin S even in the presence of inhibitors with subnanomolar inhibitory constant values. These differences were identified in cellular locations of cathepsins L and S, trafficking for secretion, co-localization with endocytosed inhibitors, and longer protein turnover time for cathepsin S compared to cathepsin L. Together, this work demonstrates that previously underappreciated cellular compensation and compartmentalization mechanisms may sustain elevated amounts of some active cathepsins while diminishing others after inhibitor treatment. This can confound predictions based solely on inhibitor kinetics, and must be better understood to effectively deploy therapies and dosing strategies that target cathepsins to prevent cancer progression., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The use of biomolecules as oxidants for the synthesis of conducting polymers provides an important tool for the control of polymer properties. Using, Pedot: PSS as a representative conducting polymer, we compare a set of heme proteins (soybean peroxidase, cytochrome c, and horseradish peroxidase) used as oxidants. The resulting, Pedot: PSS was characterized with visible and near IR spectroscopy, Fourier transform infrared spectroscopy, electron spin resonance spectroscopy, and four point probe conductivity measurements. We find that the relative concentrations of bipolarons and polarons vary as a function of the protein used for polymerization. We then show that heme degradation by hydrogen peroxide plays a critical role in determining polymer properties.

Understanding intracellular redox chemistry requires new tools for the site-specific visualization of intracellular oxidation. We have developed a spatially-resolved intracellular sensor of hydrogen peroxide, HyPer-Tau, for time-resolved imaging in live cells. This sensor consists of a hydrogen peroxide-sensing protein tethered to microtubules. We demonstrate the use of the HyPer-Tau sensor for three applications; dose-dependent response of human cells to exogenous hydrogen peroxide, a model immune response of mouse macrophages to stimulation by bacterial toxin, and a spatially-resolved response to localized delivery of hydrogen peroxide. These results demonstrate that HyPer-Tau can be used as an effective tool for tracking changes in spatially localized intracellular hydrogen peroxide and for future applications in redox signaling.

Hemoglobin- and catalase-polymerized, Pedot: PSS were characterized by X-ray photoelectron spectroscopy, visible and near-IR spectroscopy, FTIR, and ESR. Hemoglobin-polymerized, Pedot: PSS possesses bipolarons, while catalase-polymerized, Pedot: PSS is dominated by polarons. Use of heme-bound iron as an oxidant yields, Pedot: PSS with conductivity of 19.5 S cm(-1) in a single-step aqueous reaction.

All cells generate an electrical potential across their plasma membrane driven by a concentration gradient of charged ions. A typical resting membrane potential ranges from -40 to -70 mV, with a net negative charge on the cytosolic side of the membrane. Maintenance of the resting membrane potential depends on the presence of two-pore-domain potassium "leak" channels, which allow for outward diffusion of potassium ions along their concentration gradient. Disruption of the ion gradient causes the membrane potential to become more positive or more negative relative to the resting state, referred to as "depolarization" or "hyperpolarization," respectively. Changes in membrane potential have proven to be pivotal, not only in normal cell cycle progression but also in malignant transformation and tissue regeneration. Using polystyrene nanoparticles as a model system, we use flow cytometry and fluorescence microscopy to measure changes in membrane potential in response to nanoparticle binding to the plasma membrane. We find that nanoparticles with amine-modified surfaces lead to significant depolarization of both CHO and HeLa cells. In comparison, carboxylate-modified nanoparticles do not cause depolarization. Mechanistic studies suggest that this nanoparticle-induced depolarization is the result of a physical blockage of the ion channels. These experiments show that nanoparticles can alter the biological system of interest in subtle, yet important, ways.

Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular receptors used by the protein-nanoparticle complexes. These results show that protein secondary structure is a key parameter in determining the cell surface receptor used by a protein-nanoparticle complex. We expect this link between protein structure and cellular outcomes will provide a molecular basis for the design of nanoparticles for use in biological and biomedical applications.

The use of nanoparticles (NPs) in biology and medicine requires a molecular-level understanding of how NPs interact with cells in a physiological environment. A critical difference between well-controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins. It has been established that extracellular serum proteins present in blood will adsorb onto the surface of NPs, forming a "protein corona". Our goal was to understand how this protein layer affected cellular-level events, including NP binding, internalization, and transport. A combination of microscopy, which provides spatial resolution, and spectroscopy, which provides molecular information, is necessary to probe protein-NP-cell interactions. Initial experiments used a model system composed of polystyrene NPs functionalized with either amine or carboxylate groups to provide a cationic or anionic surface, respectively. Serum proteins adsorb onto the surface of both cationic and anionic NPs, forming a net anionic protein-NP complex. Although these protein-NP complexes have similar diameters and effective surface charges, they show the exact opposite behavior in terms of cellular binding. In the presence of bovine serum albumin (BSA), the cellular binding of BSA-NP complexes formed from cationic NPs is enhanced, whereas the cellular binding of BSA-NP complexes formed from anionic NPs is inhibited. These trends are independent of NP diameter or cell type. Similar results were obtained for anionic quantum dots and colloidal gold nanospheres. Using competition assays, we determined that BSA-NP complexes formed from anionic NPs bind to albumin receptors on the cell surface. BSA-NP complexes formed from cationic NPs are redirected to scavenger receptors. The observation that similar NPs with identical protein corona compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein may be responsible for the differences in cellular binding of the protein-NP complexes. Circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy show that the structure of BSA is altered following incubation with cationic NPs, but not anionic NPs. Single-particle-tracking fluorescence microscopy was used to follow the cellular internalization and transport of protein-NP complexes. The single particle-tracking experiments show that the protein corona remains bound to the NP throughout endocytic uptake and transport. The interaction of protein-NP complexes with cells is a challenging question, as the adsorbed protein corona controls the interaction of the NP with the cell; however, the NP itself alters the structure of the adsorbed protein. A combination of microscopy and spectroscopy is necessary to understand this complex interaction, enabling the rational design of NPs for biological and medical applications.

Context: Abdominal sacrocolpopexy (ASC) represents the superior treatment for apical pelvic organ prolapse (POP) but is associated with increased length of stay, analgesic requirement, and cost compared with transvaginal procedures. Laparoscopic sacrocolpopexy (LSC) and robot-assisted sacrocolpopexy (RSC) may offer shorter postoperative recovery while maintaining equivalent rates of cure., Objective: This review evaluates the literature on LSC and RSC for clinical outcomes and complications., Evidence Acquisition: A PubMed search of the available literature from 1966 to 2013 on LSC and RSC with a follow-up of at least 12 mo was performed. A total of 256 articles were screened, 69 articles selected, and outcomes from 26 presented. A review, not meta-analysis, was conducted due to the quality of the articles., Evidence Synthesis: LSC has become a mature technique with results from 11 patient series encompassing 1221 patients with a mean follow-up of 26 mo. Mean operative time was 124 min (range: 55-185) with a 3% (range: 0-11%) conversion rate. Objective cure was achieved in 91% of patients, with similar satisfaction rates (92%). Six patient series encompassing 363 patients treated with RSC with a mean follow-up of 28 mo have been reported. Mean operative time was 202 min (range: 161-288) with a 1% (range: 0-4%) conversion rate. Objective cure rate was 94%, with a 95% subjective success rate. Overall, early outcomes and complication rates for both LSC and RSC appeared comparable with open ASC., Conclusions: LSC and RSC provide excellent short- to medium-term reconstructive outcomes for patients with POP. RSC is more expensive than LSC. Further studies are required to better understand the clinical performance of RSC versus LSC and confirm long-term efficacy., Patient Summary: Laparoscopic and robot-assisted sacrocolpopexy represent attractive minimally invasive alternatives to abdominal sacrocolpopexy. They may offer reduced patient morbidity but are associated with higher costs., (Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter.

Superparamagnetic iron oxide nanoparticles (SPIONs) used as MRI contrast agents or for theranostic applications encounter a complex mixture of extracellular proteins that adsorb on the SPION surface forming a protein corona. Our goal was to understand how cellular binding and T 2 relaxation times are affected by this protein corona. Our studies focused on carboxymethyl dextran-modified SPIONs, chosen for their similarity to Resovist SPIONs used to detect liver lesions. Using a combination of fluorescence microscopy and flow cytometry, we find that the cellular binding of SPIONs to both macrophages and epithelial cells is significantly inhibited by serum proteins. To determine if this decreased binding is due to the iron oxide core or the carboxymethyl dextran surface coating, we functionalized polystyrene nanoparticles with a similar carboxymethyl dextran coating. We find a comparable decrease in cellular binding for the carboxymethyl dextran-polystyrene nanoparticles indicating that the carbohydrate surface modification is the key factor in SPION-cell interactions. NMR measurements showed that T 2 relaxation times are not affected by corona formation. These results indicate that SPIONs have a decreased binding to cells under physiological conditions, possibly limiting their use in theranostic applications. We expect these results will be useful in the design of SPIONs for future diagnostic and therapeutic applications.

Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

The use of nanoparticles for cellular therapeutic or sensing applications requires nanoparticles to bind, or adhere, to the cell surface. While nanoparticle parameters such as size, shape, charge, and composition are important factors in cellular binding, the cell itself must also be considered. All cells have an electrical potential across the plasma membrane driven by an ion gradient. Under standard conditions the ion gradient will result in a -10 to -100 mV potential across the membrane with a net negative charge on the cytosolic face. Using a combination of flow cytometry and fluorescence microscopy experiments and dissipative particle dynamics simulations, we have found that a decrease in membrane potential leads to decreased cellular binding of anionic nanoparticles. The decreased cellular binding of anionic nanoparticles is a general phenomenon, independent of depolarization method, nanoparticle composition, and cell type. Increased membrane potential reverses this trend resulting in increased binding of anionic nanoparticles. The cellular binding of cationic nanoparticles is minimally affected by membrane potential due to the interaction of cationic nanoparticles with cell surface proteins. The influence of membrane potential on the cellular binding of nanoparticles is especially important when considering the use of nanoparticles in the treatment or detection of diseases, such as cancer, in which the membrane potential is decreased.

Nuclear factor erythroid 2-related factor-2 (Nrf2) is a redox-sensitive transcription factor that activates several antioxidant and cytoprotective genes in response to oxidative stress. The role of Nrf2 activators and the intracellular regulation of Nrf2 have been studied extensively. In comparison, little is known about the self-regulation of Nrf2 due to experimental techniques commonly used to synchronize cellular signaling. Here we report that endogenous Nrf2 was downregulated in the nucleus of HeLa and MDA-MB-231 cells serum starved for 24hrs. Nrf2 expression was rescued by the addition of unconditioned media irrespective of its serum content. No concomitant change was observed in the expression of the primary inhibitor of Nrf2, Kelch-like ECH-associated protein-1 (Keap1). Nrf2 was upregulated by tert -butyl hydroquinone, although there was limited increase in Nrf2 in conditioned media-treated cells as compared to unconditioned media-treated cells. Decreasing the fraction of conditioned media in culture resulted in a dose-dependent increase in Nrf2 protein level. Taken together, our data suggests the existence of a complex self-regulatory mechanism for endogenous Nrf2 signaling.

Quantum dots (QDs) and other nanoparticles require delivery and targeting for most intracellular applications. Despite many advances, intracellular delivery and targeting remains inefficient with many QDs remaining bound to the plasma membrane rather than internalized into the cell. The fluorescence resulting from these extracellular QDs results in a background signal that competes with intracellular QDs of interest. We present two methods for the reduction and discrimination of signal resulting from plasma membrane-bound QDs. The first method, a photophysical approach, uses an extracellular quencher to greatly reduce the fluorescence signal from extracellular QDs. This method is compatible with fast, widefield, fluorescence imaging in live cells. Results are presented for two extracellular quenchers, QSY-21 and trypan blue, used in combination with 655 nm emitting QDs. The use of an extracellular quencher can be extended to a wide variety of fluorophores. The second method uses transmission electron microscopy (TEM) to image thin (60-70 nm) slices of resin-embedded cells. The use of sectioned cells and high-resolution TEM makes it possible to discriminate between plasma membrane-bound and intracellular QDs. To overcome the difficulties associated with using TEM to image individual QDs in cells, we have utilized a silver enhancement method that significantly improves the contrast of QDs in TEM images.

Purpose: We determined the feasibility of conducting a randomized clinical trial designed to compare 2 methods of manual therapy (myofascial physical therapy and global therapeutic massage) in patients with urological chronic pelvic pain syndromes., Materials and Methods: We recruited 48 subjects with chronic prostatitis/chronic pelvic pain syndrome or interstitial cystitis/painful bladder syndrome at 6 clinical centers. Eligible patients were randomized to myofascial physical therapy or global therapeutic massage and were scheduled to receive up to 10 weekly treatments of 1 hour each. Criteria to assess feasibility included adherence of therapists to prescribed therapeutic protocol as determined by records of treatment, adverse events during study treatment and rate of response to therapy as assessed by the patient global response assessment. Primary outcome analysis compared response rates between treatment arms using Mantel-Haenszel methods., Results: There were 23 (49%) men and 24 (51%) women randomized during a 6-month period. Of the patients 24 (51%) were randomized to global therapeutic massage, 23 (49%) to myofascial physical therapy and 44 (94%) completed the study. Therapist adherence to the treatment protocols was excellent. The global response assessment response rate of 57% in the myofascial physical therapy group was significantly higher than the rate of 21% in the global therapeutic massage treatment group (p = 0.03)., Conclusions: We judged the feasibility of conducting a full-scale trial of physical therapy methods and the preliminary findings of a beneficial effect of myofascial physical therapy warrants further study., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The overlap between interstitial cystitis-painful bladder syndrome and overactive bladder can present a diagnostic challenge to the treating practitioner. Both can present with similar patient symptoms. Further compounding this dilemma is the fact that no gold standard test exists to differentiate one from the other. This review will serve to highlight their similarities and distinguishing features.