Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.     

Flow patterns around ciliated microorganisms and in ciliated ducts

Microscopic organisms and tracts of ciliated epithelia often generate complicated flow patterns such as eddies or jet-like phenomena. In addition, the presence of boundaries (e.g. microscope slide or coverslip) may also influence or severely restrict the resulting fluid motion. This paper develops simplified theoretical models of flow patterns due to microorganisms, or tracts of ciliated epithelia, to help enhance our understanding of the mechanisms creating these patterns. It is demonstrated that an active driving mechanism (such as flagella or cilia) in close proximity to a non-active region (e.g. inactive cilia, non-ciliated region, slide) can produce these complicated patterns.     

MicroRNA-based silencing of Delta/Notch signaling promotes multiple cilia formation

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. Their apical surface is constituted by hundreds of motile cilia, which beat in a coordinated manner to generate directional fluid flow. We recently reported the identification of microRNAs of the miR-449 family as evolutionary conserved key regulators of vertebrate multiciliogenesis. This novel function of miR-449 was established using in vivo and in vitro antisense approaches in two distinct experimental models. miR-449 strongly accumulated in multiciliated cells in human airway epithelium and Xenopus laevis embryonic epidermis, where it triggered centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. Our data complement previous reports that showed the blocking action of miR-449 on the cell cycle, and unraveled a novel conserved mechanism whereby Notch signaling must undergo microRNA-mediated inhibition to permit differentiation of ciliated cell progenitors. We review here several important questions regarding the links between microRNAs and the Notch pathway in the control of cell fate.     

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.     

Towards the molecular dissection of fertilization signaling: Our functional genomic/proteomic strategies

Recent advances in DNA sequencing techniques and automated informatics has led to clarification of all genome sequence of some model organisms in a very short period. The demonstration of the first draft sequence of the human genome has prompted us to elaborate new approaches in biology, pharmacology and medicine. Such new research will focus on high throughput methods to function on collections of genes, and hopefully, on a genome-wide, quantitative modeling of the cell system as a whole. In this review article, we discuss the present status of "post genome sequencing" approaches in line with our strategies for understanding the molecular mechanism of fertilization and activation of development using the African clawed frog, Xenopus laevis, as a model system.     

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments, to provide an overview of progress in this rapidly-developing area of cell and developmental biology.     

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

Xenopus leads the way: Frogs as a pioneering model to understand the human brain

From its long history in the field of embryology to its recent advances in genetics, Xenopus has been an indispensable model for understanding the human brain. Foundational studies that gave us our first insights into major embryonic patterning events serve as a crucial backdrop for newer avenues of investigation into organogenesis and organ function. The vast array of tools available in Xenopus laevis and Xenopus tropicalis allows interrogation of developmental phenomena at all levels, from the molecular to the behavioral, and the application of CRISPR technology has enabled the investigation of human disorder risk genes in a higher‐throughput manner. As the only major tetrapod model in which all developmental stages are easily manipulated and observed, frogs provide the unique opportunity to study organ development from the earliest stages. All of these features make Xenopus a premier model for studying the development of the brain, a notoriously complex process that demands an understanding of all stages from fertilization to organogenesis and beyond. Importantly, core processes of brain development are conserved between Xenopus and human, underlining the advantages of this model. This review begins by summarizing discoveries made in amphibians that form the cornerstones of vertebrate neurodevelopmental biology and goes on to discuss recent advances that have catapulted our understanding of brain development in Xenopus and in relation to human development and disease. As we engage in a new era of patient‐driven gene discovery, Xenopus offers exceptional potential to uncover conserved biology underlying human brain disorders and move towards rational drug design.     

Large-scale biophysics: ion flows and regeneration

Regeneration requires exquisite orchestration of growth and morphogenesis. A powerful but still largely mysterious system of biophysical signals functions during regeneration, embryonic development and neoplasm. Ion transporters generate pH and voltage gradients, as well as ion fluxes, regulating proliferation, differentiation and migration. Endogenous bioelectrical signals are implicated in the control of wound healing, limb development, left-right patterning and spinal cord regeneration. Recent advances in molecular biology and imaging technology have allowed unprecedented insight into the sources and downstream consequences of ion flows. In complement to the current focus on molecular genetics and stem cell biology, artificial modulation of bioelectrical signals in somatic tissues is a powerful modality that might result in profound advances in understanding and augmentation of regenerative capacity.     

Nucleoplasmin: the archetypal molecular chaperone

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.     

Candida morphogenesis and host-pathogen interactions

The human fungal pathogen Candida albicans has many morphological forms. Recent advances in genomics and cell biology are providing an improved understanding of the molecular regulation of cell shape, and providing insights into the relationships between morphogenesis and virulence. This understanding may improve our ability to develop strategies to combat Candida infections.     

Histomorphology of cervical and uterine epithelia of crab-eating macaque, Macaca fasciularis.

Cytological characteristics and pattern of distribution of different cell types in the epithelia of cervix and uterus of crab-eating macaque (Macaca fascicularis) in follicular and luteal phases of the menstrual cyclic and amenorrhea were studied. The cervix uteri and uterus exhibit remarkable structural differenes in the ciliated, secretory, and ciliated-secretory cells. Since the number of ciliated-sexretory cells in the uterus is higher than in the cervix. It is believed that they form an additional source for the secretion of uterine fluid during the menstrual cylce. Both ciliated and secretory cells undergo degeneration; extensive cytoplasmic vacuolation associated with pycnosis and disorganization of the nuclei encountered.     

The molecular aspects of absorption and metabolism of carotenoids and retinoids in vertebrates

Vitamin A is an essential nutrient necessary for numerous basic physiological functions, including reproduction and development, immune cell differentiation and communication, as well as the perception of light. To evade the dire consequences of vitamin A deficiency, vertebrates have evolved specialized metabolic pathways that enable the absorption, transport, and storage of vitamin A acquired from dietary sources as preformed retinoids or provitamin A carotenoids. This evolutionary advantage requires a complex interplay between numerous specialized retinoid-transport proteins, receptors, and enzymes. Recent advances in molecular and structural biology resulted in a rapid expansion of our understanding of these processes at the molecular level. This progress opened new avenues for the therapeutic manipulation of retinoid homeostasis. In this review, we summarize current research related to the biochemistry of carotenoid and retinoid-processing proteins with special emphasis on the structural aspects of their physiological actions. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Molecular biology and gene transfer in atherosclerosis in the stenting era

Atherosclerosis is the major cause of death in the developed world. Understanding the pathogenesis of atherosclerosis has been a major challenge to cardiovascular research over the past several decades. During this period a number of advances in various scientific disciplines has increased our understanding of this disease. These include improved understanding of the structural and functional components of normal vessel wall and more recently the use of cell biology and molecular biology techniques to elucidate the pathogenesis of atherosclerosis. None of these advances has been more dramatic nor has potentially more far reaching consequences as the application of molecular biology and gene technology to the practice of cardiovascular medicine. These developments have already opened new and exciting areas of vascular research and may in the future provide for earlier identification of genetic predisposition to atherosclerosis, strategic planning of preventive therapy and more tailored pharmacologic approaches for established disease.     

Aquaporins as targets of pharmacological plant-derived compounds

Aquaporins (AQPs) are integral membrane proteins that serve as selective pores through which water and small solutes cross the plasma membranes of many human tissue and cell types. They have been identified in epithelia and endothelia involved in fluid transport, such as kidney tubules and glandular epithelia, glial cells, epidermis, and adipocytes. The pathophysiological roles of these proteins and the primary and secondary involvement of AQPs are becoming apparent in diverse clinical disorders, from diabetes insipidus to various forms of edema. The advanced understanding of aquaporin biology, from the structural determinants of channel permeability to the assignment of their physiological function in different organs, will allow the use of AQPs as targets for the therapy of a wide array of diseases. In this review, the mode of action of clinically-effective plant formulae on human AQPs-related diseases at the molecular, cellular, and organism levels is explored. The use of pharmacological plant-derived compounds as a possible strategy in the therapy of diseases related to altered water homeostasis should stimulate debate and further research objectives.     

There's more than one frog in the pond: a survey of the Amphibia and their contributions to developmental biology

The study of developmental biology has benefited greatly from the insights gained using amphibians as experimental models. Although Xenopus is currently the predominant model, much of our embryological knowledge derives from research on other amphibians. I will review some of these discoveries, made through astute choice of model organism, and I will examine the reasons behind the adoption of Xenopus as the standard for amphibian research. Additionally, I will discuss the diversity in developmental and reproductive strategies that exists within the Amphibia, and consider some of the recent advances in our understanding of the mechanisms underlying this developmental diversity.     

Offerings from an urchin

There is a natural curiosity about how organisms give rise to offspring like themselves through a series of reproducible developmental events and how, once mature, these offspring mate and continue the process giving rise the next generation. In the mid-1800 s investigators started using gametes and embryos to explore this process. Although the observations and experimental approaches changed over time, embryologists and developmental biologists after them, sought understanding of development and inheritance through the study of gametes and embryos. It is argued here that in their quests to understand these processes embryologists made major conceptual advances that were seminal to the origins of genetics and to the origins of molecular biology. Furthermore these advances derived from the distinct perspective of those investigators with focused interest on the development of the organism. In this essay fundamental discoveries that originated with the sea urchin embryo as an experimental system are used to illustrate this position. The sea urchin has a long and uninterrupted history as a model organism that helped prepare the ground for the emergence of genetics and contributed important aspects to understanding of the central dogma of molecular biology. As molecular biology came of age new concepts and technology of the discipline were transformative for developmental biology and to this day the reciprocal inductive interactions between molecular biology and developmental biology continue to revitalize each other.     

Translational systems biology of inflammation

Inflammation is a complex, multi-scale biologic response to stress that is also required for repair and regeneration after injury. Despite the repository of detailed data about the cellular and molecular processes involved in inflammation, including some understanding of its pathophysiology, little progress has been made in treating the severe inflammatory syndrome of sepsis. To address the gap between basic science knowledge and therapy for sepsis, a community of biologists and physicians is using systems biology approaches in hopes of yielding basic insights into the biology of inflammation. “Systems biology” is a discipline that combines experimental discovery with mathematical modeling to aid in the understanding of the dynamic global organization and function of a biologic system (cell to organ to organism). We propose the term translational systems biology for the application of similar tools and engineering principles to biologic systems with the primary goal of optimizing clinical practice. We describe the efforts to use translational systems biology to develop an integrated framework to gain insight into the problem of acute inflammation. Progress in understanding inflammation using translational systems biology tools highlights the promise of this multidisciplinary field. Future advances in understanding complex medical problems are highly dependent on methodological advances and integration of the computational systems biology community with biologists and clinicians.     

New insights on the molecular and cell biology of human cholangiopathies

Cholangiopathies are diseases of high social impact representing the main indication for liver transplantation in the infanthood and the third in adulthood. Despite the heterogeneous etiology and pathogenesis, cholangiopathies share many different common morphological features and, chronically progress toward a ductupenic condition clinically evidenced by the classical features of a cholestatic syndrome. The primary target of damage in the course of cholangiopathies are cholangiocytes, the epithelia cells lining the biliary tree. A bulk of researches performed in the last decade, highlighted the extraordinary biological properties of cholangiocytes involved in a number of important processes such as bile formation, proliferation, injury repair, fibrosis, angiogenesis and regulation of blood flow. Recent advances on the molecular and cell biology of human cholangiopathies are opening new potential therapeutic perspectives for these diseases.     

Gene silencing, cell fate and nuclear organisation

Although advances in molecular biology have allowed us to identify and describe many of the events associated with turning genes on, much less attention has generally been focussed on the related process of gene silencing. This is surprising as heritable gene inactivation plays an important role in determining cell lineage fates during development and defining their temporal order. Recent advances in the area of chromatin and chromosome organisation may have an impact on our understanding of cellular differentiation.