The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

The RNA-binding protein LIN28 is known to regulate cell fate, tissue growth, and pluripotency; however, a unified understanding of its role at the cellular level has not been achieved. Here, we address its developmental activity in mammalian postnatal neurogenesis. Constitutive expression of LIN28 in progenitor cells of the mouse subventricular zone (SVZ) caused several distinct effects: 1) the number of differentiated neurons in the olfactory bulb was dramatically reduced, whereas the relative abundance of 2 neuronal subtypes was significantly altered, 2) the population of proliferating neural progenitors in the SVZ was reduced, whereas the proportion of neuroblasts was increased, and 3) the number of astrocytes was reduced, occasionally causing them to appear early. Thus, LIN28 acts at a poststem cell/predifferentiation step, and its continuous expression caused a precocious phenotype unlike in other experimental systems. Furthermore, for the first time in a vertebrate system, we separate the majority of the biologic role of LIN28 from its known activity of blocking the microRNA let-7 by using a circular RNA sponge. We find that although LIN28 has a multifaceted role in the number and types of cells produced during postnatal neurogenesis, it appears that its action through let-7 is responsible for only a fraction of these effects.-Romer-Seibert, J. S., Hartman, N. W., Moss, E. G. The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

Modifications induced by chemical skin allergens on the metabolome of reconstructed human epidermis: A pilot high-resolution magic angle spinning nuclear magnetic resonance study.

BACKGROUND: High-resolution magic angle spinning (HRMAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. OBJECTIVES: To assess the impact of chemical skin sensitizers vs non-sensitizers on the metabolome of three-dimensional reconstructed human epidermis (RHE) by HRMAS NMR. METHODS: Based on the SENS-IS assay, 12 skin sensitizers and five non-sensitizing chemicals were investigated and applied on EpiSkin RHE at the published maximal non-irritating concentrations under the conditions of the test. The metabolome of RHE samples was then analyzed by HRMAS NMR. RESULTS: A total of 32 different metabolites were identified; 20 of these were quantified for all samples. Statistical univariate analysis showed that the tissue content of most measured metabolites (with the exception of acetate and glucose) was different in the untreated, treated with non-sensitizers, and treated with sensitizers samples. In RHE samples in contact with sensitizing chemicals, concentrations of 18 metabolites were significantly decreased. Alanine and tyrosine could not discriminate between sensitizer- and non-sensitizer-treated groups. A multivariate partial least-squares-discriminant analysis was performed on the two treated groups, discriminating sensitizing and non-sensitizing chemicals with a very good R2Y value of 0.87 and a good Q2Y value of 0.70. CONCLUSIONS: Data suggest that HRMAS NMR could be used to monitor the impact of chemicals, skin allergens vs non-sensitizers, on the metabolome of three-dimensional RHE.

Lin28 promotes the proliferative capacity of neural progenitor cells in brain development.

Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development.

In Situ Metabolism of Cinnamyl Alcohol in Reconstructed Human Epidermis: New Insights into the Activation of This Fragrance Skin Sensitizer.

Chemical modification of epidermal proteins by skin sensitizers is the molecular event which initiates the induction of contact allergy. However, not all chemical skin allergens react directly as haptens with epidermal proteins but need either a chemical (prehaptens) or metabolic (prohaptens) activation step to become reactive. Cinnamyl alcohol has been considered a model prohapten, as this skin sensitizer has no intrinsic reactivity. Therefore, the prevailing theory is that cinnamyl alcohol is enzymatically oxidized into the protein-reactive cinnamaldehyde, which is the sensitizing agent. Knowing that reconstructed human epidermis (RHE) models have been demonstrated to be quite similar to the normal human epidermis in terms of metabolic enzymes, use of RHE may be useful to investigate the in situ metabolism/activation of cinnamyl alcohol, particularly when coupled with high-resolution magic angle spinning nuclear magnetic resonance. Incubation of carbon-13 substituted cinnamyl derivatives with RHE did not result in the formation of cinnamaldehyde. The metabolites formed suggest the formation of an epoxy-alcohol and an allylic sulfate as potential electrophiles. These data suggest that cinnamyl alcohol is inducing skin sensitization through a route independent of the one involving cinnamaldehyde and should therefore be considered as a skin sensitizer on its own.

Ionomycin causes susceptibility to phospholipase A2 while temperature-induced increases in membrane fluidity fail: possible involvement of actin fragmentation.

A diminution in the order of membrane lipids, which occurs during apoptosis, has been shown to correlate with increased membrane susceptibility to hydrolysis by secretory phospholipase A2. Studies with artificial membranes, however, have demonstrated that the relationship between membrane order and hydrolysis is more complex than suggested thus far by cell studies. To better resolve this relationship, this study focused on comparisons between increasing temperature and calcium ionophore as means of decreasing membrane order in S49 cells. Although these two treatments caused comparable changes in apparent membrane order as detected by steady-state fluorescence measurements, only ionophore treatment enhanced phospholipase activity. Experiments with exogenously-added phosphatidylserine indicated that the difference was not due to the presence of that anionic phospholipid in the outer membrane leaflet. Instead, analysis of the equilibration kinetics of various cationic membrane probes revealed that the difference could relate to the spacing of membrane lipids. Specifically, ionophore treatment increased that spacing while temperature only affected overall membrane order and fluidity. To consider the possibility that the distinction with ionophore might relate to the actin cytoskeleton, cells were stained with phalloidin and imaged via confocal microscopy. Ionophore caused disruption of actin fibers while increased temperature did not. This apparent connection between membrane hydrolysis and the cytoskeleton was further corroborated by examining the relationship among these events during apoptosis stimulated by thapsigargin.

lin-28 controls the succession of cell fate choices via two distinct activities.

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

Lin28a regulates germ cell pool size and fertility.

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males, the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild-type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.

Fetal deficiency of lin28 programs life-long aberrations in growth and glucose metabolism.

LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here, we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO could be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.

A temporal sequence of heterochronic gene activities promotes stage-specific developmental events in Caenorhabditis elegans.

The heterochronic genes of the nematode Caenorhabditis elegans control the succession of postembryonic developmental events. The 4 core heterochronic genes lin-14, lin-28, hbl-1, and lin-41 act in a sequence to specify cell fates specific to each of the 4 larval stages. It was previously shown that lin-14 has 2 activities separated in time that promote L1 and L2 developmental events, respectively. Using the auxin-inducible degron system, we find that lin-28 and hbl-1 each have 2 activities that control L2 and L3 events which are also separated in time. Relative to events they control, both lin-28 and hbl-1 appear to act just prior to or concurrently with events of the L2. Relative to each other, lin-28 and hbl-1 appear to act simultaneously. By contrast, the lin-14 activity controlling L2 events precedes those of lin-28 and hbl-1 controlling the same events, suggesting that lin-14's regulation of lin-28 is responsible for the delay. Likewise, the activities of lin-28 and hbl-1 controlling L3 fates act well in advance of those fates, suggesting a similar regulatory gap. lin-41 acts early in the L3 to affect fates of the L4, although it was not possible to determine whether it too has 2 temporally separated activities. We also uncovered a feedback phenomenon that prevents the reactivation of heterochronic gene activity late in development after it has been downregulated. This study places the heterochronic gene activities into a timeline of postembryonic development relative to one another and to the developmental events whose timing they control.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro.

LIN28 is an RNA-binding protein that is expressed in many developing tissues. It can block let-7 (Mirlet7) microRNA processing and help promote pluripotency. We have observed LIN28 expression in the developing mouse neural tube, colocalizing with SOX2, suggesting a role in neural development. To better understand its normal developmental function, we investigated LIN28 activity during neurogliogenesis in vitro, where the succession of neuronal to glial cell fates occurs as it does in vivo. LIN28 expression was high in undifferentiated cells, and was downregulated rapidly upon differentiation. Constitutive LIN28 expression caused a complete block of gliogenesis and an increase in neurogenesis. LIN28 expression was compatible with neuronal differentiation and did not increase proliferation. LIN28 caused significant changes in gene expression prior to any effect on let-7, notably on Igf2. Furthermore, a mutant LIN28 that permitted let-7 accumulation was still able to completely block gliogenesis. Thus, at least two biological activities of LIN28 are genetically separable and might involve distinct mechanisms. LIN28 can differentially promote and inhibit specific fates and does not function exclusively by blocking let-7 family microRNAs. Importantly, the role of LIN28 in cell fate succession in vertebrate cells is analogous to its activity as a developmental timing regulator in C. elegans.

Orthologs of the Caenorhabditis elegans heterochronic genes have divergent functions in Caenorhabditis briggsae.

The heterochronic genes of Caenorhabditis elegans comprise the best-studied pathway controlling the timing of tissue and organ formation in an animal. To begin to understand the evolution of this pathway and the significance of the relationships among its components, we characterized 11 Caenorhabditis briggsae orthologs of C. elegans heterochronic genes. Using CRISPR/Cas9, we made a variety of alleles and found that several mutant phenotypes differ in significant ways from those of C. elegans. Although most mutant orthologs displayed defects in developmental timing, their phenotypes could differ in which stages were affected, the penetrance and expressivity of the phenotypes, or by having additional pleiotropies that were not obviously connected to developmental timing. However, when examining pairwise epistasis and synergistic relationships, we found those paralleled the known relationships between their C. elegans orthologs, suggesting that the arrangements of these genes in functional modules are conserved, but the modules' relationships to each other and/or to their targets has drifted since the time of the species' last common ancestor. Furthermore, our investigation has revealed a relationship between this pathway to other aspects of the animal's growth and development, including gonad development, which is relevant to both species.

The Attending of the Day ("The Pretending"): An Exercise in Autonomy.

Background As regulations governing appropriate resident supervision increase, it has become increasingly difficult to provide residents with the appropriate level of autonomy during their training years. The "Attending of the Day" describes an experiential teaching method that provides a balance between learners' autonomy and appropriate supervision. Methodology Each day one member of the inpatient team is selected as the "Attending of the Day," or "The Pretending." She or he then performs the typical duties of the teaching faculty, from medical decision-making regarding patient care to educating other team members during rounds. "The Pretending" is directly supervised by the clinical faculty. Results Using the grounded theory methodology, we analyzed 935 anonymous evaluations from students and residents over 14 years, leading to the identification of the following three major themes: created an enabling learning environment, provided autonomy, and improved confidence. These results led to the inclusion of the technique as part of the Back to Bedside initiative, which was rated as an essential tool in building confidence and autonomy by 75% of the participants in the 2018 Accreditation Council for Graduate Medical Education's Back to Bedside residents' well-being survey. Recently, the Jacobs School of Medicine launched the Moments of Excellence in Education: Recognition and Inspiration (MEE:RI) program which gives students a way to recognize exemplary moments of teaching they encounter. The "Attending of the Day" method received recognition as a transformative experience in students' medical education. Conclusions The "Attending of the Day" is the first innovative experiential learning technique that allows learners of all levels in both Undergraduate Medical Education (UME) and Graduate Medical Education (GME) to practice and assess autonomy. This innovation suggests that residents and students are looking for opportunities to challenge themselves. "The Pretending" allows them to experience those challenges in an empowering learning environment while they gradually build their confidence on the path to achieving progressive autonomy.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

Heterochronic genes and the nature of developmental time.

Timing is a fundamental issue in development, with a range of implications from birth defects to evolution. In the roundworm Caenorhabditis elegans, the heterochronic genes encode components of a molecular developmental timing mechanism. This mechanism functions in diverse cell types throughout the animal to specify cell fates at each larval stage. MicroRNAs play an important role in this mechanism by stage-specifically repressing cell-fate regulators. Recent studies reveal the surprising complexity surrounding this regulation--for example, a positive feedback loop may make the regulation more robust, and certain components of the mechanism are expressed in brief periods at each stage. Other factors reveal the potential for important roles of steroid hormones and targeted proteolysis. Investigation of the heterochronic genes has revealed a mechanism composed of precisely timed switches linked to discrete developmental stages. Timing is a dimension of developmental regulation that may be difficult to witness in vertebrates because developmental stages are not as discrete as in C. elegans, each tissue is likely to be independently regulated. Homologs of certain heterochronic genes of vertebrates show temporally regulated expression patterns, and may ultimately reveal timing mechanisms not previously known to exist.

Experimental melting curve of zirconium metal to 37 GPa.

In this report, we present results of high-pressure experiments probing the melt line of zirconium (Zr) up to 37 GPa. This investigation has determined that temperature versus laser power curves provide an accurate method to determine melt temperatures. When this information is combined with the onset of diffuse scattering, which is associated with the melt process, we demonstrate the ability to accurately determine the melt boundary. This presents a reliable method for rapid determination of melt boundary and agrees well with other established techniques for such measurements, as reported in previous works on Zr.

Room-temperature compression and equation of state of body-centered cubic zirconium.

Zirconium (Zr) has properties conducive to nuclear applications and exhibits complex behavior at high pressure with respect to the effects of impurities, deviatoric stress, kinetics, and grain growth which makes it scientifically interesting. Here, we present experimental results on the 300 K equation of state of ultra-high purity Zr obtained using the diamond-anvil cell coupled with synchrotron-based x-ray diffraction and electrical resistance measurements. Based on quasi-hydrostatic room-temperature compression in helium to pressure P = 69.4(2) GPa, we constrain the bulk modulus and its pressure derivative of body-centered cubic (bcc) ß-Zr to be K = 224(2) GPa and K' = 2.6(1) at P = 37.0(1) GPa. A Monte Carlo approach was developed to accurately quantify the uncertainties in K and K'. In the Monte Carlo simulations, both the unit-cell volume and pressure vary according to their experimental uncertainty. Our high-pressure studies do not indicate additional isostructural volume collapse in the bcc phase of Zr in the 56-58 GPa pressure range.

The place of psychodynamic psychotherapy in the integrated treatment of posttraumatic stress disorder and trauma recovery.

Psychodynamic psychotherapists treating posttraumatic stress disorder (PTSD) sufferers can draw on an accumulated body of trauma studies from their own field to guide their work. However, these reports, often based on case studies or conceptual reviews, do not have the same empirical conclusiveness as more recent evidence-based research demonstrating the efficacy of cognitive-behavioral and body-oriented therapies. In this article, a psychodynamic psychotherapist reflects on his treatment of an Israeli man who developed PTSD after enduring 4 terrorist attacks. The author shows how assimilative integration offered him a theory- and research-based model that helped him comfortably combine separate treatment interventions. He also shows how this model helped him locate with some precision the specific contribution of psychodynamic psychotherapy. (PsycINFO Database Record (c) 2010 APA, all rights reserved).

The holding/containment function in supervision groups for group therapists.

Group supervision is essential for group therapists, given the difficulty of following and reflecting on the multiplicity of processes in therapy groups and the unique opportunity of exploring them as they resonate and become enacted in the supervision group. More specifically, the paper discusses the critical function of holding/containment in supervision groups. Clinical vignettes are presented to highlight the importance of the holding/containment function and the obstacles that hinder its emergence. The role of the therapist's interpretations as a primary means for ensuring effective holding and containment is examined.

MicroRNAs: hidden in the genome.

Genes for tiny RNAs have been found to be plentiful in the genomes of worms, flies, humans and probably all animals. Some of these microRNAs have been conserved through evolution, and many are expressed only at specific times or places. How they act is just beginning to be understood, but their importance to biology is likely to be great.