A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

Technical Considerations in the Freezing, Low-Temperature Storage and Thawing of Stem Cells for Cellular Therapies.

The commercial and clinical development of cellular therapy products will invariably require cryopreservation and frozen storage of cellular starting materials, intermediates and/or final product. Optimising cryopreservation is as important as optimisation of the cell culture process in obtaining maximum yield and a consistent end-product. Suboptimal cryopreservation can lead not only to batch-to-batch variation, lowered cellular functionality and reduced cell yield, but also to the potential selection of subpopulations with genetic or epigenetic characteristics divergent from the original cell line. Regulatory requirements also impact on cryopreservation as these will require a robust and reproducible approach to the freezing, storage and thawing of the product. This requires attention to all aspects of the application of low temperatures: from the choice of freezing container and cryoprotectant, the cooling rate employed and its mode of de-livery, the correct handling of the frozen material during storage and transportation, to the eventual thawing of the product by the end-user. Each of these influences all of the others to a greater or lesser extent and none should be ignored. This paper seeks to provide practical insights and alternative solutions to the technical challenges faced during cryopreservation of cells for use in cellular therapies.

Energy efficiency and color quality limits in artificial light sources emulating natural illumination.

We present in this work a calculation of the theoretical limits attainable for natural light emulation with regard to the joint optimization of the Luminous Efficacy of Radiation and color fidelity by using multiple reflectance spectra datasets, along with an implementation of a physical device that approaches these limits. A reduced visible spectrum of blackbody radiators is introduced and demonstrated which allows lamps designed to emulate natural light to operate with excellent color fidelity and higher efficiency as compared to full visible spectrum sources. It is shown that even though 3,000K and 5,500K blackbody sources have maximum efficacies of 21 lm/W and 89 lm/W, respectively, reduced-spectrum artificial light sources can exceed those values up to 363 lm/W and 313 lm/W, respectively, while retaining excellent color fidelity. Experimental demonstration approaching these values is accomplished through the design and implementation of a 12-channel light engine which emits arbitrarily-tunable spectra. The color fidelity of the designed spectra is assessed through Color Rendering Maps, showing that color fidelity is preserved uniformly over a large spectral reflectance dataset, unlike other approaches to generate white light.

Participatory community action research in homeless shelters: Outcomes for shelter residents and service-learning research assistants.

The article reports empirical outcomes of an ongoing transdisciplinary participatory community action research project that implements behavioral activation in homeless shelters. The overall goal of this Project is twofold: (1) to improve psychosocial functioning of shelter residents and enhance their opportunities to overcome homelessness; and (2) to enhance civic development of service-learning students who assist in Project implementation. Two studies are reported, representing these goals. Study 1 found that residents of a men's shelter (n = 892), women's shelter (n = 433), and transitional housing (n = 40) perceived behavioral activation sessions as immediately beneficial (i.e., important, meaningful, worthy of repeating, and enjoyable), and over the course of shelter stay, they perceived behavioral activation as contributing to their hope, empowerment/self-sufficiency, quality of life, purpose/meaning in life, wellbeing, social support, shelter social climate, and relationships with staff. Quantitative findings are supported by qualitative data (comments by residents on forms). Study 2, which replicates and extends past research on civic-development in service-learning students, used a new quasi-experimental design to compare service-learning students (n = 41) in an interdisciplinary course on homelessness versus non-service-learning students (n = 16) in a psychology course. Service-learning students showed pre- to post-semester improvements in community service self-efficacy, decreases in stigmatizing attitudes, and increases in awareness of privilege and oppression, but students not engaged in service-learning did not show these civic-related changes. These quantitative results are supported by qualitative data (written reflections by students). Results and implications are discussed within the context of the concept of psychopolitical validity.

Color rendering map: a graphical metric for assessment of illumination.

The method of evaluating color rendering using a visual, graphical metric is presented. A two-dimensional Color Rendering Map (CRM) of a light source's color-rendering capabilities is explained and demonstrated. Extension of this technique to three-dimensional CRMs of objects under illumination is explained, including the method of introducing numerical indices in order to evaluate standards for specific applications in lighting. Three diverse applications, having a range from subtle to significant color variation, are shown with their respective CRMs. These three applications are also used to demonstrate how three differing light sources produce different maps. The results show a flexible, simple method to obtain a clear, visual determination of color rendering performance from differing sources used in differing illumination applications. The use of numeric indices in these applications shows how specific standards can be imposed in assessing the applicability of a light source.

De-entangling colorfulness and fidelity for a complete statistical description of color quality.

In this Letter, the main attributes known to affect color quality are treated statistically over a set of 118 spectra representing the current mainstream lighting technology. The color rendering index (CRI) is used to assess color fidelity while colorfulness is used to complement CRI-R(a), supported by the growing evidence that assessment of light spectra cannot overlook color preference inputs. Colorfulness is evaluated by our optimal color (O(c)) index, through a code that computes the (MacAdam) theoretical maximum volumetric gamut of objects under a given illuminant for all the spectra in our database. Pearson correlation coefficients for CRI-R(a), the (Y. Ohno's) color quality scale (CQS) and O(c) show a high correlation (0.950) between CRI-R(a) and CQS-Q(a), while O(c) shows the lowest correlation (0.577) with CRI-R(a), meaning that O(c) represents the best complement to CRI-R(a) and Q(a) for an in-depth study of color quality.

Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif.

Proneural proteins play a central role in vertebrate neurogenesis, but little is known of the genes that they regulate and of the factors that interact with proneural proteins to activate a neurogenic program. Here, we demonstrate that the proneural protein Mash1 and the POU proteins Brn1 and Brn2 interact on the promoter of the Notch ligand Delta1 and synergistically activate Delta1 transcription, a key step in neurogenesis. Overexpression experiments in vivo indicate that Brn2, like Mash1, regulates additional aspects of neurogenesis, including the division of progenitors and the differentiation and migration of neurons. We identify by in silico screening a number of additional candidate target genes, which are recognized by Mash1 and Brn proteins through a DNA-binding motif similar to that found in the Delta1 gene and present a broad range of activities. We thus propose that Mash1 synergizes with Brn factors to regulate multiple steps of neurogenesis.

Metal-nitride-oxide-semiconductor light-emitting devices for general lighting.

The potential for application of silicon nitride-based light sources to general lighting is reported. The mechanism of current injection and transport in silicon nitride layers and silicon oxide tunnel layers is determined by electro-optical characterization of both bi- and tri-layers. It is shown that red luminescence is due to bipolar injection by direct tunneling, whereas Poole-Frenkel ionization is responsible for blue-green emission. The emission appears warm white to the eye, and the technology has potential for large-area lighting devices. A photometric study, including color rendering, color quality and luminous efficacy of radiation, measured under various AC excitation conditions, is given for a spectrum deemed promising for lighting. A correlated color temperature of 4800K was obtained using a 35% duty cycle of the AC excitation signal. Under these conditions, values for general color rendering index of 93 and luminous efficacy of radiation of 112 lm/W are demonstrated. This proof of concept demonstrates that mature silicon technology, which is extendable to low-cost, large-area lamps, can be used for general lighting purposes. Once the external quantum efficiency is improved to exceed 10%, this technique could be competitive with other energy-efficient solid-state lighting options.

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes.

The bHLH (basic helix-loop-helix) transcription factor Mash1 is best known for its role in the regulation of neurogenesis. However, Mash1 is also expressed in oligodendrocyte precursors and has recently been shown to promote the generation of oligodendrocytes in cell culture, suggesting that it may regulate oligodendrogenesis as well. Here, we show that in the developing ventral forebrain, Mash1 is expressed by a subset of oligodendrocyte precursors (OPCs) as soon as they are generated in the ventricular zone. Using reporter mice, we demonstrate that a subset of OPCs in both the embryonic and postnatal forebrain originate from Mash1-positive progenitors, including a large fraction of adult NG2-positive OPCs. Using Mash1 null mutant mice, we show that Mash1 is required for the generation of an early population of OPCs in the ventral forebrain between embryonic day 11.5 (E11.5) and E13.5, whereas OPCs generated later in embryonic development are not affected. Overexpression of Mash1 in the dorsal telencephalon induces expression of PDGFRalpha (platelet-derived growth factor receptor alpha) but not other OPC markers, suggesting that Mash1 specifies oligodendrogenesis in cooperation with other factors. Analysis of double-mutant mice suggests that Olig2 is one of the factors that cooperate with Mash1 for generation of OPCs. Together, our results show for the first time that Mash1 cooperates in vivo with Olig2 in oligodendrocyte specification, demonstrating an essential role for Mash1 in the generation of a subset of oligodendrocytes and revealing a genetic heterogeneity of oligodendrocyte lineages in the mouse forebrain.

Severe Parechovirus 3 Infections in Young Infants-Kansas and Missouri, 2014.

BACKGROUND: Infection with parechovirus type 3 (PeV3) can cause severe neurologic and sepsis-like illness in young infants; clinical and epidemiologic descriptions have been limited. We aimed to characterize PeV3 illness and explore risk factors for acquisition in a cluster of neonatal cases at Children's Mercy Hospital in Kansas City, Missouri. METHODS: Cerebrospinal fluid specimens were obtained from infants aged <180 days who were hospitalized with sepsis-like illness or meningitis between June 1 and November 1, 2014. PeV-positive specimens were sequenced at the Centers for Disease Control and Prevention. We reviewed the medical and birth charts of the infants and performed face-to-face parent interviews. We analyzed characteristics according to infant age and intensive care admission status. RESULTS: We identified 35 cases of PeV infection in infants aged 5 to 56 days. Seven infants required intensive care (median age, 11 days vs 27 days among those who did not require intensive care; P = .0044). Six of these 7 infants had neurologic manifestations consistent with seizures, and all 6 of them were treated with acyclovir but subsequently tested negative for herpes simplex virus. Virus sequences formed 2 lineages, both of which were associated with severe illness. Half of the infants were reported to have household contacts who were ill during the week before onset. Infants aged ≤7 days at onset were more likely to have been delivered at the same hospital. CONCLUSIONS: PeV3 can cause severe neurologic illness in neonates, and younger infants are more likely to require intensive care. PeV3 should be considered along with herpes simplex virus and other pathogens when evaluating young infants with sepsis-like illness or meningitis. More widespread testing for PeV3 would enable us to gain a better understanding of the clinical scope and circulation of this virus.

Cryopreservation of human embryonic stem cell lines.

Two different approaches have been adopted for the cryopreservation of human embryonic stem cells (hESCs): vitrification and conventional slow cooling/rapid warming. The vitrification method described here is designed for hESCs that grow as discrete colonies on a feeder cell monolayer, and are subcultured by manual subdivision of the colonies into multicellular clumps. hESCs that are subcultured by enzymatic dissociation can more conveniently be cryopreserved by conventional slow cooling/rapid warming methods. Although both methods are suitable for use in a research context, neither is suitable for cryopreservation of embryonic stem cells destined for clinical diagnostic or therapeutic uses without modification.

Evaluation of postinjury hepatocyte function by central amino acid clearance.

It has been demonstrated by other investigators that central plasma clearance of amino acids accurately predicts hepatocyte function in patients with liver disease and correlates with clinical outcome. This methodology has not heretofore been studied in the trauma patient. It is our hypothesis that central amino acid clearance in trauma patients is more reflective of hepatocyte function than traditional liver function tests. We examined the plasma amino acid clearance rates using L-[1-13C]phenylalanine. Clearance rates were compared to standard liver function tests (LFTs) and the sensitivity and predictability of the technique were determined. The study was conducted on uninjured control subjects and in seriously injured patients, both with and without significant liver injuries. Compared to baseline values in the control group, initial phenylalanine breath scores were reduced in the injured, but exceeded control levels at 7 days postinjury. These changes were statistically significant. There was no difference between those with and without liver trauma. LFTs showed inconsistent and conflicting results. Thus, central amino acid clearance measured by L-[1-13C]phenylalanine oxidation is depressed immediately following injury but reaches supranormal levels at 7 days postinjury. Compared to LFTs, amino acid clearance suggests initial hepatocyte suppression followed by hyperactivity and is a more accurate determinant of hepatocyte function.

Cryopreservation: Vitrification and Controlled Rate Cooling.

Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.

Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies.

Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1-2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.

Preservation and stability of cell therapy products: recommendations from an expert workshop.

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.

Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood.

Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50 percent. In the context of umbilical cord blood such losses are unacceptable. Whilst an empirical approach can provide an acceptable level of recovery, the cryopreservation process can only be optimised by adopting a methodological approach. This paper provides an overview of just such an approach as illustrated by a study on CD34 cells from umbilical cord blood. It involves firstly the determination of membrane permeability parameters that can then be used to model safe addition and elution protocols for the chosen cryoprotectant, in this case dimethyl sulphoxide. This in turn permits cryoprotectant toxicity to be evaluated free from the confounding effect of osmotic damage caused by inappropriate addition and elution protocols. Finally, non-toxic concentrations of cryoprotectant may be investigated in a cooling rate study to provide an optimal cryopreservation protocol. Using the model, the effect on CD34 cells of current addition and elution protocols was also examined.

Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.

Quiescence is essential for long-term maintenance of adult stem cells. Niche signals regulate the transit of stem cells from dormant to activated states. Here, we show that the E3-ubiquitin ligase Huwe1 (HECT, UBA, and WWE domain-containing 1) is required for proliferating stem cells of the adult mouse hippocampus to return to quiescence. Huwe1 destabilizes proactivation protein Ascl1 (achaete-scute family bHLH transcription factor 1) in proliferating hippocampal stem cells, which prevents accumulation of cyclin Ds and promotes the return to a resting state. When stem cells fail to return to quiescence, the proliferative stem cell pool becomes depleted. Thus, long-term maintenance of hippocampal neurogenesis depends on the return of stem cells to a transient quiescent state through the rapid degradation of a key proactivation factor.

The UK Stem Cell Bank: its role as a public research resource centre providing access to well-characterised seed stocks of human stem cell lines.

The rapidly expanding field of stem cell research offers the potential to develop therapeutic agents to treat diseases such as Parkinson's, diabetes and heart disease. It is important that stem cell lines derived from quality-controlled and well-characterised cell banks should be made available to both the scientific and clinical communities to promote high-quality research and development. The requirement in the United Kingdom (UK) for rigorous regulation of the procurement and use of embryonic stem (ES) cell lines led the UK government to fund the establishment of a national bank for stem cell lines. The UK Stem Cell Bank (UKSCB) hosted at the National Institute for Biological Standards and Control (NIBSC) is committed to working closely with the clinical and research communities to provide qualified stocks of human stem cell lines of adult, foetal and embryonic origin for both research use and for use in emerging human therapy.

The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.