Nuclear DNA and protein content evaluation in Taxus plant cell cultures using multiparameter flow cytometry

Plant cell cultures of Taxus provide the most reliable production methods for the anti-cancer drug paclitaxel. In order to comprehend the inherent culture heterogeneity and production variability in cell cultures, it is essential that the cellular metabolism is studied at the genomic level. Genomic stability in plant cell cultures is crucial as it affects cell growth and division, metabolite accumulation and protein synthesis. A rapid and efficient method to prepare nuclei suspensions from aggregated cell cultures of Taxus was employed. Methods were subsequently developed to simultaneously stain them for DNA and protein content using Propidium Iodide and Fluorescein Isothiocyanate respectively. Flow cytometry was used to analyze and quantify the DNA content and genome size of Taxus using known reference species as standards. Furthermore, their genomic stability was evaluated by correlating DNA content and genome size with cell size and complexity, protein content, and elicitation effects using multiparameter flow cytometry. These techniques to evaluate and correlate various culture characteristics can be very useful in designing superior bio processes for enhanced production.


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Drafting and Implementing an Effective Social Media Policy

Social media is everywhere and used in many business and personal situations. There is no indication that social media use is declining; rather, social media use is constantly expanding into new realms and taking on new forms. Social media launches political campaigns, international pop stars, and new businesses to heightened levels of success or failure with just a few mouse clicks. Because social media information has the ability to spread rapidly, not addressing social media or hoping it will not affect the employer\u27s business is a dangerous practice. Currently, few employers have a Social Media Policy ( Policy ). By not having a Policy, the employer and its business are left vulnerable to the whims of its employees\u27 social media actions and cannot guide employees toward using social media to protect and further the employer\u27s business purpose. A Policy\u27s existence makes the employer proactive rather than reactive. Further, establishing a Policy provides employees with clear expectations about when social media can be used, for what purposes, and what level of privacy an employee should expect regarding personal use. Finally, the employer needs a Policy to promote consistent enforcement among all employees. Inconsistent actions by supervisors for similar employee actions could open the employer up to potential employment discrimination lawsuits. Once the employer understands that it needs a Policy, the next step is deciding what type to implement. The employer must create a Policy that furthers the employer\u27s business purposes. Regardless of whether the employer decides to ban social media or freely allow its use, the employer should always spell it out in a Policy that defines the parameters of social media use. However, before the employer establishes a Policy it should conduct a cost-benefit analysis of social media uses and benefits for the employer versus the cost and resources required to enforce such a Policy. The employer should consider the Policy\u27s monitoring and enforcement costs and the employee productivity costs depending on the level of allowed social media use. Taking into account these considerations, this Article will instruct an employer or employer\u27s counsel on how to best draft an effective Policy that will meet all of the employer\u27s objectives

Early Childhood Teacher Turnover in Nebraska

Teacher turnover is a serious challenge across early childhood settings. Turnover can be expensive for early childhood programs, burdensome to staff, and harmful to children throughout the nation. Nebraska is no exception. This research brief describes teacher turnover in the state’s early care and education settings, including licensed child care, state-funded PreK, and Kindergarten through Grade 3. Research Questions The following research questions were asked across early childhood programs (licensed child care, state-funded PreK, and K-3): 1. What was the average rate of annual teacher turnover? 2. According to administrators, what was the most common reason teachers left their employment? 3. Which hiring challenges did administrators experience in filling positions? 4. On average

Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

BACKGROUND: Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype. METHODS: To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later. RESULTS: Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model. CONCLUSIONS: These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.R01 AR060217 - NIAMS NIH HHS; R01 AR060209 - NIAMS NIH HHS; T32 HD043730 - NICHD NIH HHS; R00 HL098453 - NHLBI NIH HHS; R00HL098453 - NHLBI NIH HHS; R01AR060209 - NIAMS NIH HHS; R01AR060217 - NIAMS NIH HH

Regulating repression : roles for the Sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

The Gasser laboratory is supported by the Novartis Research Foundation and the EU training network Nucleosome 4D. SK was supported by an EMBO long-term fellowship, a Schrodinger fellowship from the FWF, and the Swiss SystemsX.ch initiative/C-CINA; HCF by an EMBO long-term fellowship.Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.Publisher PDFPeer reviewe

Workforce well-being: Personal and workplace contributions to early educators\u27 depression across settings

Building on research demonstrating the importance of teachers\u27 well-being, this study examined personal and contextual factors related to early childhood educators\u27 (n =1640) depressive symptoms across licensed child care homes, centers, and schools. Aspects of teachers\u27 beliefs, economic status, and work-related stress were explored, and components of each emerged as significant in an OLS regression. After controlling for demographics and setting, teachers with more adult-centered beliefs, lower wages, multiple jobs, no health insurance, more workplace demands, and fewer work-related resources, had more depressive symptoms. Adult-centered beliefs were more closely associated with depression for teachers working in home-based settings compared to center-based settings. These findings provide preliminary evidence about what relates to depression in the early childhood workforce, which has implications for supporting well-being across settings

Risk Factors for Depression Among Early Childhood Teachers

This study examined possible risk factors associated with teachers’ depression in a variety of early childhood settings. Teachers with lower pay, no health insurance, multiple jobs, greater job stress, and more adult-centered beliefs reported more symptoms of depression. To reduce these symptoms, efforts should be made to support teachers’ mental health at multiple levels, including individual, environmental, and policy. Researchers used data collected in 2015-16 from a large survey of early childhood educators in Nebraska. Four early childhood settings were sampled: licensed family child care homes (home-based), licensed child care centers (center-based), state-funded PreK programs, and elementary schools serving children in Kindergarten through Grade 3 (K-3). Across settings, a total of 1,640 teachers responded to the survey: 36% in K-3, 25% home-based, 23% center-based, and 17% PreK. The survey included various measures, including economic circumstances (health insurance status, pay, public assistance use, and working multiple jobs), work-related stress, beliefs about children’s development (the extent to which teachers held more adult-centered vs. child-centered beliefs), and symptoms of depression

Treatment of the lung injury of drowning: a systematic review

Background: Drowning is a cause of significant global mortality. The mechanism of injury involves inhalation of water, lung injury and hypoxia. This systematic review addressed the following question: In drowning patients with lung injury, what is the evidence from primary studies regarding treatment strategies and subsequent patient outcomes? Methods: The search strategy utilised PRISMA guidelines. Databases searched were MEDLINE, EMBASE, CINAHL, Web of Science and SCOPUS. There were no restrictions on publication date or age of participants. Quality of evidence was evaluated using GRADE methodology. Results: Forty-one papers were included. The quality of evidence was very low. Seventeen papers addressed the lung injury of drowning in their research question and 24 had less specific research questions, however included relevant outcome data. There were 21 studies regarding extra-corporeal life support, 14 papers covering the theme of ventilation strategies, 14 addressed antibiotic use, seven papers addressed steroid use and five studies investigating diuretic use. There were no clinical trials. One retrospective comparison of therapeutic strategies was found. There was insufficient evidence to make recommendations as to best practice when supplemental oxygen alone is insufficient. Mechanical ventilation is associated with barotrauma in drowning patients, but the evidence predates the practice of lung protective ventilation. There was insufficient evidence to make recommendations regarding adjuvant therapies. Conclusions: Treating the lung injury of drowning has a limited evidentiary basis. There is an urgent need for comparative studies of therapeutic strategies in drowning

Genetic and environmental influences on eating behavior - a study of twin pairs reared apart or reared together

This study examined the relative influence of genetic versus environmental factors on specific aspects of eating behavior. Adult monozygotic twins (22 pairs and 3 singleton reared apart, 38 pairs and 9 singleton reared together, age 18-76 years, BMI 17-43 kg/m2) completed the Three Factor Eating Questionnaire. Genetic and environmental variance components were determined for the three eating behavior constructs and their subscales using model-fitting univariate and multivariate analyses. Unique environmental factors had a substantial influence on all eating behavior variables (explaining 45-71% of variance), and most strongly influenced external locus for hunger and strategic dieting behavior of restraint (explaining 71% and 69% of variance, respectively). Genetic factors had a statistically significant influence on only 4 variables: restraint, emotional susceptibility to disinhibition, situational susceptibility to disinhibition, and internal locus for hunger (heritabilities were 52%, 55%, 38% and 50%, respectively). Common environmental factors did not statistically significantly influence any variable assessed in this study. In addition, multivariate analyses showed that disinhibition and hunger share a common influence, while restraint appears to be a distinct construct. These findings suggest that the majority of variation in eating behavior variables is associated with unique environmental factors, and highlights the importance of the environment in facilitating specific eating behaviors that may promote excess weight gain.R01 AR046124 - NIAMS NIH HHS; R01 MH065322 - NIMH NIH HHS; T32 HL069772 - NHLBI NIH HHS; R37 DA018673 - NIDA NIH HHS; R01 DK073321 - NIDDK NIH HHS; R01 DA018673 - NIDA NIH HH

Perceptions of Familial Risk in those Seeking a Genetic Risk Assessment for Alzheimer’s Disease

Perceived risk is a complex concept that influences the genetic counseling process and can affect client coping and behavior. Although the association between family history and risk perception is well recognized in the literature, no studies have explored this relationship specifically in those seeking genetic susceptibility testing for a common chronic condition. REVEAL is a randomized trial assessing the impact of APOE disclosure and genetic risk assessment for Alzheimer’s disease (AD). Using baseline REVEAL data, we hypothesized that there would be a significant association between the degree of AD family history and risk perception of AD, and that this relationship would be stronger in those who believed that genetics is a very important AD risk factor. In our sample of 293 participants, we found that a higher self‐perceived risk of AD was associated with strength of family history of AD (p < 0.001), belief in genetics as an important AD risk factor (p < 0.001), being female (p < 0.001) and being Caucasian (p = 0.02). These results are the first to demonstrate the association between family history and risk perception in persons volunteering for genetic susceptibility testing for a common complex disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147109/1/jgc40130.pd