Cyclin B2 is required for progression through meiosis in mouse oocytes.

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity.

To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in Dictyostelium and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.

Early Indicators of NCLEX-RN performance.

Health Education Systems Incorporated (HESI(™)) test results, course grades, and National Council Licensure Examination-Registered Nurse (NCLEX-RN(®)) outcomes of students in an associate degree nursing program at a midwestern public university were investigated. Statistical analysis revealed that introductory Fundamentals HESI test scores, more than either comprehensive HESI Exit Exam scores or other specialty HESI test scores, significantly predicted NCLEX-RN outcomes in this study (p < 0.05), while controlling for grade point average and high school percentile rank. In addition, of the general education courses and the nursing courses in the associate nursing program examined, Pediatric Nursing, Medical-Surgical Nursing, and Maternity Nursing course grades were found most statistically significantly influential of all the HESI test scores (p < 0.01).

FLN-2 functions in parallel to LINC complexes and Cdc42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and pro-inflammatory responses. Studies performed in tissue culture cells have implicated LINC (linker of nucleoskeleton and cytoskeleton) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In C. elegans larvae, 6 pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function, this and structural predictions suggest that FLN-2 is not a divergent filamin. The immunoglobulin (Ig)-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.

In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific to Ras, Rac or Rho small GTPases of the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components of signal transduction networks which link upstream G-protein coupled receptors to downstream cytoskeletal components and help cells migrate through their dynamic microenvironment. Conventional approaches to perturb and examine these signaling and cytoskeletal networks, such as gene knockout or overexpression, are protracted which allows networks to readjust through gene expression changes. Moreover, these tools lack spatial resolution to probe the effects of local network activations. To overcome these challenges, blue light-inducible cryptochrome- and LOV domain-based dimerization systems have been recently developed to control signaling or cytoskeletal events in a spatiotemporally precise manner. We illustrate that, within minutes of global membrane recruitment of full-length GEFs or their catalytic domains only, widespread increases or decreases in F-actin rich protrusions and cell size occur, depending on the particular node in the networks targeted. Additionally, we demonstrate localized GEF recruitment as a robust assay system to study local network activation-driven changes in polarity and directed migration. Altogether, these optical tools confirmed GEFs of Ras superfamily GTPases as regulators of cell shape, actin dynamics, and polarity. Furthermore, this optogenetic toolbox may be exploited in perturbing complex signaling interactions in varied physiological contexts including mammalian embryogenesis.

[Exploring the Interplay between communication of the Gospel und popular religion. A case study on the church-launched YouTube channel "Jana believes"]. / Kommunikation des Evangeliums und Populäre Religion. Annäherung an ein Spannungsverhältnis am Beispiel des YouTube-Kanals "Jana glaubt".

The relationship between religion and popular culture has attracted considerable attention in the field of the sociology of religion. However, church-based religious communication has rarely been explored in this perspective. This in mind, the paper explores the YouTube channel "Jana believes", a pioneering and also controversial project sponsored by the Evangelical Church in Germany.The argumentation proceeds in five steps. First, Hubert Knoblauch's concept of "popular religion" is interlinked with the practical-theological perspective of "communicating the Gospel" as well as with more recent approaches to the mediatization of religion. Then characteristic elements and video genres of YouTube communication as part of popular culture are explicated. Against this background, the distinct profile of the YouTube channel "Jana believes" is outlined, with specific regard to the vlog genre. Subsequently, the interplay between popular religion, communicating the Gospel and mediatization of religion is explored in a more in depth-analysis of two exemplary video sequences.Finally, the summary brings together both sides of the interplay in question: On the one hand, the channel videos are designed according to established standards of personalized YouTube communication. In line with the concept of popular religion, the boundaries between private and public become fluid. Public communication of the Gospel is constituted by sharing what is private in life and faith. On the other hand, in the case of this channel the subjective enactment of faith is also shaped and transformed by the involvement of the church. Particularly regarding questions of authenticity and authority, the tension between popular religion and communicating the Gospel at times leads to potentially conflictual negotiation processes.

Using Live-Cell Imaging and Synthetic Biology to Probe Directed Migration in Dictyostelium.

For decades, the social amoeba Dictyostelium discoideum has been an invaluable tool for dissecting the biology of eukaryotic cells. Its short growth cycle and genetic tractability make it ideal for a variety of biochemical, cell biological, and biophysical assays. Dictyostelium have been widely used as a model of eukaryotic cell motility because the signaling and mechanical networks which they use to steer and produce forward motion are highly conserved. Because these migration networks consist of hundreds of interconnected proteins, perturbing individual molecules can have subtle effects or alter cell morphology and signaling in major unpredictable ways. Therefore, to fully understand this network, we must be able to quantitatively assess the consequences of abrupt modifications. This ability will allow us better control cell migration, which is critical for development and disease, in vivo. Here, we review recent advances in imaging, synthetic biology, and computational analysis which enable researchers to tune the activity of individual molecules in single living cells and precisely measure the effects on cellular motility and signaling. We also provide practical advice and resources to assist in applying these approaches in Dictyostelium.

Pretest online discussion groups to augment teaching and learning.

Tests and final examination scores of three semesters of control students in a nursing foundation course were compared with tests and final examination scores of three semesters of participating students. Participating students were offered access to an asynchronous pretest online discussion activity with a faculty e-moderator. While the simplified Bloom's revised taxonomy assisted in creating appropriate preparatory test and final examination questions for pretest online discussion, Salmon's five-stage online method provided direction to the e-moderator on how to encourage students to achieve Bloom's higher-order thinking skills during the pretest online discussions. Statistical analysis showed the pretest online discussion activity had a generally positive impact on tests and final examination scores, when controlling for a number of possible confounding variables, including instructor, cumulative grade point average, age, and credit hours.

Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner.

The spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. Each mammalian kinetochore binds many microtubules, but how many attached microtubules are required to turn off the checkpoint, and how the kinetochore monitors microtubule numbers, are not known and are central to understanding SAC mechanisms and function. To address these questions, here we systematically tune and fix the fraction of Hec1 molecules capable of microtubule binding. We show that Hec1 molecules independently bind microtubules within single kinetochores, but that the kinetochore does not independently process attachment information from different molecules. Few attached microtubules (20% occupancy) can trigger complete Mad1 loss, and Mad1 loss is slower in this case. Finally, we show using laser ablation that individual kinetochores detect changes in microtubule binding, not in spindle forces that accompany attachment. Thus, the mammalian kinetochore responds specifically to the binding of each microtubule and counts microtubules as a single unit in a sensitive and switch-like manner. This may allow kinetochores to rapidly react to early attachments and maintain a robust SAC response despite dynamic microtubule numbers.

The mammalian kinetochore-microtubule interface: robust mechanics and computation with many microtubules.

The kinetochore drives chromosome segregation at cell division. It acts as a physical link between chromosomes and dynamic microtubules, and as a signaling hub detecting and processing microtubule attachments to control anaphase onset. The mammalian kinetochore is a large macromolecular machine that forms a dynamic interface with the many microtubules that it binds. While we know most of the kinetochore's component parts, how they work together to give rise to its robust functions remains poorly understood. Here we highlight recent findings that shed light on this question, driven by an expanding physical and molecular toolkit. We present emerging principles that underlie the kinetochore's robust microtubule grip, such as redundancy, specialization, and dynamicity, and present signal processing principles that connect this microtubule grip to robust computation. Throughout, we identify open questions, and define simple engineering concepts that provide insight into kinetochore function.

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance.

Curriculum Evaluation Using Path Analysis.

The purpose of this study was to explore the application of path analysis to evaluate the curriculum model and provide guidance in course sequencing. Using statistical package R to add and subtract various path connections, the curriculum model was improved to a proposed curriculum model, which passed the exact-fit test (χ42 = 45.612, with P = .286 > .05). Path analysis provided an objective method to evaluate the curriculum model and course sequencing in the baccalaureate program under study, while informing possible placement of courses.

Detection of Salmonella by bacteriophage Felix 01.

Salmonellae are mammalian pathogens that are transmitted mainly through foodstuffs and their handlers. Rapid detection requires both specificity and sensitivity in samples containing other bacteria. A solution to this problem is the use of the great specificity conferred by bacteriophages. After implanting reporter genes in a phage genome, the reporter gene products can be measured with great sensitivity when a bacterial host is present. Bacteriophage Felix 01 infects almost all Salmonella strains and has been manipulated to contain the lux genes specifying bacterial luciferase, an enzyme that converts chemical energy to visible light. A widely applicable methodology for preventing the escape of such recombinant phage has also been developed.

Spindle assembly checkpoint satisfaction occurs via end-on but not lateral attachments under tension.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. What signals the SAC monitors remains unclear. We do not know the contributions of different microtubule attachment features or tension from biorientation to SAC satisfaction nor how these possible cues change during attachment. In this study, we quantify concurrent Mad1 intensity and report on SAC silencing, real-time attachment geometry, occupancy, and tension at individual mammalian kinetochores. We show that Mad1 loss from the kinetochore is switch-like with robust kinetics and that tension across sister kinetochores is established just before Mad1 loss events at the first sister. We demonstrate that CenpE-mediated lateral attachment of the second sister can persistently generate this metaphase-like tension before biorientation, likely stabilizing sister end-on attachment, yet cannot induce Mad1 loss from that kinetochore. Instead, Mad1 loss begins after several end-on microtubules attach. Thus, end-on attachment provides geometry-specific molecular cues or force on specific kinetochore linkages that other attachment geometries cannot provide.

The interaction of stressful life events and a serotonin transporter polymorphism in the prediction of episodes of major depression: a replication.

CONTEXT: Prior evidence from twin studies suggested genetic moderation of the depressogenic effects of stressful life events (SLEs). Can the specific genes involved in this effect be identified? OBJECTIVE: To replicate and extend a recent study that a functional variant in the serotonin transporter (5-HTT) might in part explain these findings. DESIGN: Characterizing risk for major depression and generalized anxiety syndrome in the last year as a function of 5-HTT genotype, sex, and the occurrence of SLEs and ratings of the SLE-associated level of threat. SETTING: A population-based sample of adult twins. PARTICIPANTS: Five hundred forty-nine male and female twins with a mean age at participation of 34.9 years (SD 9.1). MAIN OUTCOME MEASURE: Episodes of major depression and generalized anxiety syndrome in the last year with onset measured to the nearest month. RESULTS: Individuals with 2 short (S) alleles at the 5-HTT locus were more sensitive to the depressogenic effects of all SLEs than were those with 1 or 2 long (L) alleles. When level of SLE-associated threat was examined, the interaction between genotype and SLE resulted from an increased sensitivity of SS individuals to the depressogenic effects of common low-threat events. These events had little impact on risk for those possessing the SL and LL genotypes. The 5-HTT genotype did not modify the effects of SLEs on risk for generalized anxiety syndrome. CONCLUSION: Variation at the 5-HTT moderates the sensitivity of individuals to the depressogenic effects of SLEs largely by producing, in SS individuals, an increased sensitivity to the impact of mild stressors. Replication of these intriguing results is needed.

Visualization of active biomass distribution in a BGAC fluidized bed reactor using GFP tagged Pseudomonas putida F1.

A favorable microenvironment for biofilm growth on GAC particles was shown using green fluorescent protein (GFP) as a marker for a phenol degrading bacterium, Pseudomonas putida F1. The dispersion of P. putida F1 in a biofilm covering granulated activated carbon (GAC) particles was monitored and compared to a biofilm on non-activated granular carbon particles. Laser scanning confocal microscopy (LSCM) micrographs of the biofilms taken from two fluidized bed reactors operating under identical conditions, showed higher fluorescent green areas in the GAC biofilm, especially close to the GAC surface. Quantitative analysis of the biofilm by COMSTAT, a three-dimensional biofilm structure analysis program, showed higher biomass concentration and higher viability in the GAC covered biofilm vs. the non-activated carbon biofilm. In addition, better effluent quality was measured for the BGAC reactor, which strongly suggests a significantly larger biofilm surface area available to the substrate, as opposed to that of the non-activated carbon carrier reactor.

Childhood adversity, monoamine oxidase a genotype, and risk for conduct disorder.

BACKGROUND: Very little is known about how different sets of risk factors interact to influence risk for psychiatric disorder. OBJECTIVE: To replicate a recent report of a genotype-environment interaction that predicts risk for antisocial behavior in boys. DESIGN: Characterizing risk for conduct disorder in boys in association with monoamine oxidase A genotype and exposure to familial adversity, defined by interparental violence, parental neglect, and inconsistent discipline. SETTING: A community-based sample of twin boys. PARTICIPANTS: Five hundred fourteen male twins aged 8 to 17 years. MAIN OUTCOME MEASURE: Conduct disorder. RESULTS: There was a main effect of adversity but not of monoamine oxidase A on risk for conduct disorder. Low monoamine oxidase A activity increased risk for conduct disorder only in the presence of an adverse childhood environment. Neither a passive nor an evocative genotype-environment correlation accounted for the interaction. CONCLUSION: This study replicates a recent report of a genotype-environment interaction that predicts individual variation in risk for antisocial behavior in boys.

The interrelationship of neuroticism, sex, and stressful life events in the prediction of episodes of major depression.

OBJECTIVE: Three potent risk factors for major depression are female sex, the personality trait of neuroticism, and adversity resulting from exposure to stressful life events. Little is known about how they interrelate in the etiology of depressive illness. METHOD: In over 7,500 individual twins from a population-based sample, the authors used a Cox proportional hazard model to predict onsets of episodes of DSM-III-R major depression in the year before the latest interviews on the basis of previously assessed neuroticism, sex, and adversity during the past year; adversity was operationalized as the long-term contextual threat scored from 15 life event categories. RESULTS: In the best-fit Cox model for prediction of depressive onsets, neuroticism, female sex, and greater adversity all strongly increased risk for major depression. An interaction was seen between neuroticism and adversity such that individuals with high neuroticism were at greater overall risk for major depression and were more sensitive to the depressogenic effects of adversity. An interaction was also seen between adversity and sex, as the excess risk for major depression in women was confined to individuals with low stress exposure. CONCLUSIONS: Psychosocial adversity interacts both with neuroticism and with sex in the etiology of major depression. The impact of neuroticism on illness risk is greater at high than at low levels of adversity, while the effect of sex on probability of onset is the opposite--greater at low than at high levels of stress. Complete etiologic models for major depression should incorporate interactions between risk factor classes.

Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella.

A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.