Turnaround time reduction for military certificates of compliance - team 1

The objective of this project is to reduce the turnaround time for military Certificates of Compliance (COC) at the Milliken Company in Marietta, S.C. The first step was to determine the needs of the clients and determine the major losses of the system by performing a physical and Why-Why analysis as well as utilizing fishbone diagrams. With this information it was possible to then construct a Pareto chart to show the clients where the large percentage of losses is coming from. The team found that the key losses in the system came from having to send material to an outside lab for testing and that lab technicians allowed large losses when machines were allowed to sit idle for extended periods of time. Using this data the team will generate a set of solutions to tackle the scheduling issues that will optimize the dry-lab testing procedures

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

<p>Abstract</p> <p>Background</p> <p>Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, <it>Mollicutes</it>. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins.</p> <p>Results</p> <p>Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the <it>Mollicutes</it>. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling.</p> <p>Conclusions</p> <p>We describe novel features of PARCELs (<b>P</b>alindromic <b>A</b>mphipathic <b>R</b>epeat <b>C</b>oding <b>EL</b>ements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches.</p

Androgen regulation of axon growth and neurite extension in motoneurons

Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injurie

Th17 Cell Response in SOD1G93A Mice following Motor Nerve Injury

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4(+) T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1(G93A)). SOD1(G93A) mice, compared with WT mice, displayed an increase in the basal activation state of CD4(+) T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1(G93A) mice exhibit abnormal CD4(+) T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease

Hypothalamic Neuroendocrine Functions in Rats with Dihydrotestosterone-Induced Polycystic Ovary Syndrome: Effects of Low-Frequency Electro-Acupuncture

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5α-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4–5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS

Th17 Cell Response in SOD1 G93A Mice following Motor Nerve Injury

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4 + T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1 G93A ). SOD1 G93A mice, compared with WT mice, displayed an increase in the basal activation state of CD4 + T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1 G93A mice exhibit abnormal CD4 + T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease

Prenatal Hyperandrogenization Induces Metabolic and Endocrine Alterations Which Depend on the Levels of Testosterone Exposure

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life

Developmental programming of the female neuroendocrine system by steroids

Developmental programming refers to processes that occur during early life that may have long-term consequences, modulating adult health and disease. Complex diseases, such as diabetes, cancer and cardiovascular disease, have a high prevalence in different populations, are multifactorial, and may have a strong environmental component. The environment interacts with organisms, affecting their behaviour, morphology and physiology. This interaction may induce permanent or long-term changes, and organisms may be more susceptible to environmental factors during certain developmental stages, such as the prenatal and early postnatal periods. Several factors have been identified as responsible for inducing the reprogramming of various reproductive and nonreproductive tissues. Among them, both natural and synthetic steroids, such as endocrine disruptors, are known to have either detrimental or positive effects on organisms depending on the dose of exposure, stage of development and biological sexual background. The present review focuses on the action of steroids and endocrine disruptors as agents involved in developmental programming and on their modulation and effects on female neuroendocrine functions.Fil: Abruzzese, Giselle Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Crisosto, Nicolás. Facultad de Medicina de la Universidad de Chile; Chile. Clinica Las Condes; ChileFil: De Grava Kempinas, Wilma. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Sotomayor Zárate, Ramón. Universidad de Valparaiso; Chil

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium