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Decreased ovarian testosterone production, granulosa cell dysfunction, oocyte telomere shortening and mitochondrial defects, and sperm DNA fragmentation all contribute to reproductive aging. Maneuvers aimed at correcting these abnormalities, including reduction of oxidative stress, improved lifestyle and nutrition, and the role of supplements, are reviewed. [ABSTRACT FROM AUTHOR]

The authors review and discuss numerous factors that influence erectile function and their interactions, based on the published literature. Of critical importance are vascular nitric oxide; nutrition; exercise; weight control and maintaining insulin sensitivity; early treatment of hypertension with attention to effects on erectile function; avoiding sources of oxidative stress such as obesity and smoking; reducing inflammation (e.g. from gingivitis); improving pelvic floor muscle strength; and inhibiting cyclic GMP break-down. The described interventions act on different aspects of erectile biochemistry and physiology. Therefore, combining multiple therapeutic approaches will yield maximum benefits for erectile and vascular and general health.

A two-photon absorbing (2PA) red emitter group was chemically conjugated onto amphiphilic poly(ethylene glycol)- block -poly( ε -caprolactone) (PEG- b -PCL) copolymers, and further grafted with cyclo (Arg-Gly-Asp) (cRGD) peptide to form micelle 1. Micelle 1 with cRGD targeting groups were used for targeted bioimaging. For comparison, micelle 2 without the cRGD targeting groups were also prepared and investigated. The micelles were characterized using dynamic light scattering (DLS), showing average diameters of around 77 nm. The cRGD targeting group is known to bind specifically with α v β 3 integrin in cancer cells. In this study, α v β 3 integrin overexpressed human glioblastoma U87MG cell line and α v β 3 integrin deficient human cervical cancer HeLa cell line were chosen. Results showed that the cRGD targeting group enhanced the cellular uptake efficiency of the micelles significantly in α v β 3 integrin rich U87MG cells. Higher temperature (37 °C versus 4 °C) and calcium ions (with 3M calcium chloride in the cell culture medium versus no addition of calcium ions) enhanced the cellular uptake efficiency, suggesting that the uptake of the micelles is through the endocytosis pathway in cells. A 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay was used to evaluate the cytotoxicity of the micelles and no significant cytotoxicity was observed. The BTD-containing two-photon absorbing emitter in the micelles showed a two-photon absorbing cross-section of 236 GM (1GM = 1 × 10 -50 cm 4 s photon -1 molecule -1 ) at 820 nm, which is among the highest values reported for red 2PA emitters. Because of the two-photon absorbing characteristics, micelle 1 was successfully used for two-photon fluorescence imaging targeted to U87MG cells under a two-photon fluorescence microscope. This study is the first report regarding the targeted imaging of a specific cancer cell line (herein, U87MG) using the BTD-conjugated-fluorophore-containing block copolymers., Competing Interests: Conflicts of interest There are no conflicts to declare.

We present a new approach for three-dimensional (3D) live single-cell imaging with isotropic sub-micron spatial resolution using fluorescence computed tomography (fCT). A thin, highly inclined and laminated optical (HILO) sheet of light is used for fluorescence excitation in live single cells that are rotated around an axis perpendicular to the optical axis. During a full rotation, 400-500 two-dimensional (2D) projection images of the cell are acquired from multiple viewing perspectives by rapidly scanning the HILO light sheet along the optical axis. We report technical characteristics of the HILO approach and the results of a quantitative comparison with conventional epi fCT, demonstrating that HILO fCT offers significantly (about 17 times) reduced photobleaching and a two-fold improvement in 3D imaging contrast. We discuss potential application areas of the method for cell structure studies in live single cells with isotropic 3D spatial resolution.

This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques., (© 2018 Wiley Periodicals, Inc.)

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)-a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)-a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

Precise spatial positioning and isolation of mammalian cells is a critical component of many single cell experimental methods and biological engineering applications. Although a variety of cell patterning methods have been demonstrated, many of these methods subject cells to high stress environments, discriminate against certain phenotypes, or are a challenge to implement. Here, we demonstrate a rapid, simple, indiscriminate, and minimally perturbing cell patterning method using a laser fabricated polymer stencil. The stencil fabrication process requires no stencil-substrate alignment, and is readily adaptable to various substrate geometries and experiments.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs. We present a simple and rapid method to produce small populations with varying size of epithelial cells (10-50 cells/population) with high-throughput (~ 1 population/second) on flat surfaces via patterning of extracellular matrix (ECM) proteins and random seeding of cells. We demonstrate that despite inherent limitations of non-contact, drop-on-demand piezoelectric inkjet printing for protein patterning, varying mixtures of ECM proteins can be deposited with high reproducibility and level of control on glass substrates using a set of dynamically adjustable optimized deposition parameters. We demonstrate high consistency for the number of cells per population (~1 cell standard error of mean), the population's size (~0.2 coefficient of variation) and shape, as well as accurate spatial placement of and distance between colonies of a panel of metaplastic and dysplastic esophageal epithelial cells with differing adhesion and motility characteristics. The number of cells per colony, colony size and shape can be varied by dynamically varying the amount of ECM proteins deposited per spatial location and the number of spatial locations on the substrate. The method is applicable to a broad range of biological and biomedical studies including cell-cell communications, cellular microenvironment, migration, and stimulus response.

Women bear the predominant burden of our obesogenic environment, with a higher incidence of obesity than men, more impact on their fertility and success with treatment, and significant maternal and perinatal morbidity and mortality. In this series, the causes, consequences, and solutions regarding the obesity pandemic, the mechanisms of the effect of obesity on the female and male, the epigenetic consequences of male obesity, the marked effects on perinatal outcomes, and the effects of weight loss before conception and during pregnancy are explored. Lifestyle modifications, in particular a healthy diet and exercise during the 3-6 months before conception and during treatment, should result in better outcomes than requiring weight loss before fertility treatments. Such fundamental changes toward a healthier lifestyle will achieve steady and sustainable weight loss and long-term benefits for general health. The role of bariatric surgery before pregnancy requires careful consideration., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Obesity has become pandemic owing to an obesogenic environment (inexpensive calorie dense food, technologies and structure of communities that reduce or replace physical activity, and inexpensive nonphysical entertainment) and excessive emphasis on low fat intake resulting in excessive intake of simple carbohydrates and sugar. Effects are greater for women owing to their smaller size and extra weight gain with each pregnancy, with 38% of American adult women being obese. Women are responsible for more than three-fourths of the more than 400 billion dollars of excess direct health care expenditures due to obesity. They are less likely to conceive naturally and with fertility treatments, more likely to miscarry, and have more prematurity and other complications with their pregnancies. We describe the many causes, including key roles that a dysbiotic intestinal microbiome plays in metabolic derangements accompanying obesity, increased calorie absorption, and increased appetite and fat storage. Genetic causes are contributory if these other factors are present but have limited effect in isolation. The numerous health consequences of obesity are discussed. The authors itemize ways that an individual and societies can mitigate the pandemic. However, individual will power, the will of society to enact change, and willingness of the public to accept outside intervention frustrate efforts to stabilize or reverse this crisis. The most promising strategies are education and efforts by individuals to make responsible choices several times every day to protect, most effectively by prevention, their most valuable asset., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

This work describes an enhancement to the loop-mediated isothermal amplification (LAMP) reaction which results in improved performance. Enhancement is achieved by adding a new set of primers to conventional LAMP reactions. These primers are termed "swarm primers" based on their relatively high concentration and their ability to create new amplicons despite the theoretical lack of single-stranded annealing sites. The primers target a region upstream of the FIP/BIP primer recognition sequences on opposite strands, substantially overlapping F1/B1 sites. Thus, despite the addition of a new primer set to an already complex assay, no significant increase in assay complexity is incurred. Swarm priming is presented for three DNA templates: Lambda phage, Synechocystis sp. PCC 6803 rbcL gene, and human HFE. The results of adding swarm primers to conventional LAMP reactions include increased amplification speed, increased indicator contrast, and increased reaction products. For at least one template, minor improvements in assay repeatability are also shown. In addition, swarm priming is shown to be effective at increasing the reaction speed for RNA amplification via RT-LAMP. Collectively, these results suggest that the addition of swarm primers will likely benefit most if not all existing LAMP assays based on state-of-the-art, six-primer reactions.

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using C t value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages., (© The Author 2016. Published by Oxford University Press. All rights reserved.)

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett's esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts., Competing Interests: This work was supported by the following grants to D.R.M. from the NIH National Human Genome Research Institute, Centers of Excellence in Genomic Sciences: 5 P50HG002360 and 3P50HG002360-10S1.

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8-naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8-naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt-4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt-4 stood out as a potential mitochondrial dye for living-cell experiments involving induced mitochondrial stress arising from the treatments because Mt-4 shows enhanced fluorescence in mitochondrial stress situations., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat's differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.