An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis

Transitions in Gambling Participation During Late Adolescence and Young Adulthood

Purpose: The purpose of this study was to examine transitions in gambling participation from late adolescence into emerging adulthood and to identify factors (i.e., gender, race, intervention status, lunch status, conduct disorder, parental monitoring, neighborhood environment, and substance use) that might influence these transitions. Methods: Markov modeling was used to describe the movement between past-year gambling states (i.e., nongambling and gambling) across 5 years. Annual data on the past-year gambling behavior and substance use were collected from 515 young men and women starting at the age of 17 years. Results: Past-year gambling declined from 51% prevalence at the age of 17 years to 21% prevalence at the age of 22 years. Participants who reported no past-year gambling at a particular annual assessment had more than an 80% probability of also reporting no past-year gambling at the following assessment. Men were 1.07–2.82 times more likely than women to transition from past-year nongambling to gambling year to year, and women were 1.27–5.26 times more likely than men to transition from past-year gambling to nongambling year to year. In addition, gender and past-year tobacco use interacted such that men who used tobacco were most likely (and men who did not use tobacco least likely) to gamble at baseline. Conclusions: Transition rates between gambling states appear to be relatively stable over time from late adolescence into emerging adulthood; however, men and those who engage in substance use may be at an increased risk of gambling participation

Transitions in Gambling Participation During Late Adolescence and Young Adulthood

Purpose: The purpose of this study was to examine transitions in gambling participation from late adolescence into emerging adulthood and to identify factors (i.e., gender, race, intervention status, lunch status, conduct disorder, parental monitoring, neighborhood environment, and substance use) that might influence these transitions. Methods: Markov modeling was used to describe the movement between past-year gambling states (i.e., nongambling and gambling) across 5 years. Annual data on the past-year gambling behavior and substance use were collected from 515 young men and women starting at the age of 17 years. Results: Past-year gambling declined from 51% prevalence at the age of 17 years to 21% prevalence at the age of 22 years. Participants who reported no past-year gambling at a particular annual assessment had more than an 80% probability of also reporting no past-year gambling at the following assessment. Men were 1.07–2.82 times more likely than women to transition from past-year nongambling to gambling year to year, and women were 1.27–5.26 times more likely than men to transition from past-year gambling to nongambling year to year. In addition, gender and past-year tobacco use interacted such that men who used tobacco were most likely (and men who did not use tobacco least likely) to gamble at baseline. Conclusions: Transition rates between gambling states appear to be relatively stable over time from late adolescence into emerging adulthood; however, men and those who engage in substance use may be at an increased risk of gambling participation

A Decline in Walking Speed is Associated with Incident Knee Replacement in Adults with and at Risk for Knee Osteoarthritis

OBJECTIVE: To determine if a one-year change in walking speed is associated with receiving an incident knee replacement during the following year in adults with and at risk for knee osteoarthritis (OA). METHODS: Using data from the Osteoarthritis Initiative, we determined a one-year change in 20- meter walk speed from three observation periods (i.e., 0-12, 12-24, and 24-36 month). We operationally defined one-year change in walking speed as either: 1) decline: \u3c -0.1 m/s change, 2) no change: between -0.1 and 0.1 m/s change, 3) increase: \u3e 0.1 m/s change. Incident knee replacement was defined using each subsequent one-year period (i.e., 12-24, 24- 36, and 36-48 month). Combining data from the three observation periods, we performed a Poisson regression with robust error variance to determine the relative risk between a change in walking speed (exposure) and incident knee replacement over the following year (outcome). RESULTS: Of the 4,264 participants included within this analysis (11,311 total person visits), 115 (3%) adults received a knee replacement. Decline in walking speed was associated with a 104% increase in risk [adjusted relative risk (RR)=2.04; 95% confidence interval (CI)= 1.40-2.98], while an increase in walking speed associated with a 55% decrease in risk (RR=0.45; 95% CI=0.22-0.93) of incident knee replacement in the following year compared to a person with no change in walking speed. CONCLUSION: A one-year decline in walking speed is associated with an increased risk, while one-year increase in walking speed is associated with a decreased risk of future incident knee replacement

Self-Assembled Molecular-Electronic Films Controlled by Room Temperature Quantum Interference

If single-molecule, room-temperature, quantum interference (QI) effects could be translated into massively parallel arrays of molecules located between planar electrodes, QI-controlled molecular transistors would become available as building blocks for future electronic devices. Here, we demonstrate unequivocal signatures of room-temperature QI in vertical tunneling transistors, formed from self-assembled monolayers (SAMs), with stable room-temperature switching operations. As a result of constructive QI effects, the conductances of the junctions formed from anthanthrene-based molecules with two different connectivities differ by a factor of 34, which can further increase to 173 by controlling the molecule-electrode interface with different terminal groups. Field-effect control is achieved using an ionic liquid gate, whose strong vertical electric field penetrates through the graphene layer and tunes the energy levels of the SAMs. The resulting room-temperature on-off current ratio of the lowest-conductance SAMs can reach up to 306, about one order of magnitude higher than that of the highest-conductance SAMs

Effect of Ex Vivo Ionizing Radiation on Static and Fatigue Properties of Mouse Vertebral Bodies

For a variety of medical and scientific reasons, human bones can be exposed to a wide range of ionizing radiation levels. In vivo radiation therapy (0.05 kGy) is used in cancer treatment, and ex vivo irradiation (25-35 kGy) is used to sterilize bone allografts. Ionizing radiation in these applications has been shown to increase risk of fracture, decrease bone quality and degrade collagen integrity. Past studies have investigated the deleterious effects of radiation on cortical or trabecular bone specimens individually, but to date no studies have examined whole bones containing both cortical and trabecular tissue. Furthermore, a clear relationship between the dose and the mechanical and biochemical response of bone's extracellular matrix has yet to be established for doses ranging from cancer therapy to allograft sterilization (0.05-35 kGy). To gain insight into these issues, we conducted an ex vivo radiation study to investigate non-cellular (i.e. matrix) effects of ionizing radiation dose on vertebral whole bone mechanical properties, over a range of radiation doses (0.05-35 kGy), with a focus on any radiation-induced changes in collagen. With underlying mechanisms of action in mind, we hypothesized that any induced reductions in mechanical properties would be associated with changes in collagen integrity. METHODS: 20-week old female mice were euthanized and the lumbar spine was dissected using IACUC approved protocols. The lumbar vertebrae (L1- S1) were extracted from the spine via cuts through adjacent intervertebral discs, and the endplates, posterior processes, surrounding musculature, and soft tissues were removed (approx. 1.5mm diameter, approx. 2mm height). Specimens were randomly assigned to one of five groups for ex vivo radiation exposure: x-ray irradiation at 0.05, 1, 17, or 35 kGy, or a 0 kGy control. Following irradiation, the vertebrae were imaged using microcomputed tomography (micro-CT) and then subjected to either monotonic compressive loading to failure or uniform cyclic compressive loading. During cyclic testing, samples were loaded in force control to a force level that corresponded to a strain of 0.46%, as determined in advance by a linearly elastic micro-CT-based finite element analysis for each specimen. Tests were stopped at imminent fracture, defined as a rapid increase in strain. The main outcome for the monotonic test was the strength (maximum force); for cyclic testing it was the fatigue life (log of the number of cycles of loading at imminent failure). A fluorometric assay was used on the S1 vertebrae to measure the number of non-enzymatic collagen crosslinks[4]. A one-way ANOVA was performed on mechanical properties and collagen crosslinks; means were compared with controls using Dunnett's method, with a Tukey-Kramer post-hoc analysis when significance was found (p 0.05). The finite element analysis prescribed force level for cyclic loading exceeded the measured (monotonic) strength of the 17 and 35 kGy irradiated groups (mean +/- SD, 20.6 +/- 5.6 N; 13.2 +/- 3.7 N, respectively) and therefore these groups were eliminated from the fatigue study. The fatigue life for the 0.05 and 1 kGy groups were similar to each other and were not statistically significantly different from the control group (Figure 1c)

Ionizing Radiation from Ex Vivo Sterilization Diminishes Collagen Integrity and Vertebral Body Mechanics

Clinical exposure to ionizing radiation could put cancer radiotherapy or bone allograft patients at an increased risk of fracture. In these applications, ionizing radiation levels can range from accumulative 50 Gy for radiotherapy cancer treatment, to acute 35,000 Gy for allograft sterilization. Ionizing radiation has been shown to decrease bon equality through reduced strength and post-yield properties and degrade collagen integrity through either increased crosslinks (advanced glycation end products, AGEs)or fragmentation. It is unclear which collagen structural change accounts for reduced strength. The dose-dependent effect of ionizing radiation on mechanical and biochemical properties of whole bones are not well understood, particularly for ex vivo doses ranging from 50 to 35,000 Gy

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics

Metabolic gatekeeper function of B-lymphoid transcription factors.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation

The Functional DRD3 Ser9Gly Polymorphism (rs6280) Is Pleiotropic, Affecting Reward as Well as Movement

Abnormalities of motivation and behavior in the context of reward are a fundamental component of addiction and mood disorders. Here we test the effect of a functional missense mutation in the dopamine 3 receptor (DRD3) gene (ser9gly, rs6280) on reward-associated dopamine (DA) release in the striatum. Twenty-six healthy controls (HCs) and 10 unmedicated subjects with major depressive disorder (MDD) completed two positron emission tomography (PET) scans with [11C]raclopride using the bolus plus constant infusion method. On one occasion subjects completed a sensorimotor task (control condition) and on another occasion subjects completed a gambling task (reward condition). A linear regression analysis controlling for age, sex, diagnosis, and self-reported anhedonia indicated that during receipt of unpredictable monetary reward the glycine allele was associated with a greater reduction in D2/3 receptor binding (i.e., increased reward-related DA release) in the middle (anterior) caudate (p<0.01) and the ventral striatum (p<0.05). The possible functional effect of the ser9gly polymorphism on DA release is consistent with previous work demonstrating that the glycine allele yields D3 autoreceptors that have a higher affinity for DA and display more robust intracellular signaling. Preclinical evidence indicates that chronic stress and aversive stimulation induce activation of the DA system, raising the possibility that the glycine allele, by virtue of its facilitatory effect on striatal DA release, increases susceptibility to hyperdopaminergic responses that have previously been associated with stress, addiction, and psychosis