Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance.

Feeding is an essential biological process for an organism's growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans (C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Proteostasis is differentially modulated by inhibition of translation initiation or elongation.

Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

Impact of Technique-Specific Operative Videos on First-Year Dental Students' Performance of Restorative Procedures.

The aim of this study was to examine the impact of psychomotor operative video demonstrations on first-year dental students who are performing specific procedures for the first time in a preclinical setting. The class was randomly divided into two groups, and three restorative procedures were selected. On the date on which each procedure was to be performed in the preclinical laboratory for the first time, one group (experimental, n=50) was shown a technique video for that specific procedure immediately before commencing the exercise; the control cohort (n=50) did not view the video. Technical performance on procedures was evaluated by students and two calibrated and blinded examiners. The students' perceptions of the experience were also collected in a survey. All first-year students participated in the study, for a 100% response rate. A Mann-Whitney U test did not show any group differences in technical performance (mean values on preparation: 77.1 vs. 77.8; amalgam: 82.7 vs. 82.8; composite: 79.7 vs. 78.0). A Spearman rho test revealed a significantly higher correlation in 13 out of 25 evaluation categories between student self-assessment and blinded examiner assessment for the experimental group. A chi-square test of questionnaire responses revealed a positive student perception of administering these videos for the preparation (X(2)=4.8, p<0.03), the amalgam restoration (X(2)=12.4, p<0.001), and the composite restoration (X(2)=11.3, p<0.001). The psychomotor video demonstrations did not immediately improve student performance on preclinical operative procedures, but they were well received by students and augmented self-assessment ability. These findings suggest that videos can be a useful teaching aid in a preclinical environment, especially regarding comprehension of concepts.

Patient Engagement in Neurological Clinical Trials Design: A Conference Summary.

OBJECTIVES: The conference objectives included educating patients and advocates about clinical trials, educating the clinical research community about patient perspectives on participating in clinical trial design, and identifying strategies to increase participation in clinical trial design for neurological disorders. DESIGN: Observations were noted during a 1-day conference attended by patients, patient advocates, clinical trial staff, and investigators. The conference offered didactic sessions, small, and large group discussions. PARTICIPANTS: Conference participants were patients, patient advocates, clinical trial staff, students, and investigators interested in engaging patients in clinical trial design for neurological disorders. MEASURES: Conference participants were asked to consider lessons learned that could increase patient engagement in clinical trial design. RESULTS: We found that there is growing interest in including patients in the design of clinical trials for neurological disorders. Several themes emerged on how to move forward: networking; the multifaceted roles of advocates in research; training and education; creating patient-researcher partnerships; and clinical trials regulation issues. CONCLUSIONS: The conference provided a forum for dialogue regarding stakeholder engagement in the design of clinical trials for neurological disorders. This experience provides a template for replication and dissemination of this conference and informs next steps to accelerate the pathway from dialogue to action.

Overview, hurdles, and future work in adaptive designs: perspectives from a National Institutes of Health-funded workshop.

BACKGROUND: The clinical trials community has a never-ending search for dependable and reliable ways to improve clinical research. This exploration has led to considerable interest in adaptive clinical trial designs, which provide the flexibility to adjust trial characteristics on the basis of data reviewed at interim stages. Statisticians and clinical investigators have proposed or implemented a wide variety of adaptations in clinical trials, but specific approaches have met with differing levels of support. Within industry, investigators are actively exploring the benefits and pitfalls associated with adaptive designs (ADs). For example, a Drug Information Association (DIA) working group on ADs has engaged regulatory agencies in discussions. Many researchers working on publicly funded clinical trials, however, are not yet fully engaged in this discussion. We organized the Scientific Advances in Adaptive Clinical Trial Designs Workshop to begin a conversation about using ADs in publicly funded research. Held in November of 2009, the 1½-day workshop brought together representatives from the National Institutes of Health (NIH), the Food and Drug Administration (FDA), the European Medicines Agency (EMA), the pharmaceutical industry, nonprofit foundations, the patient advocacy community, and academia. The workshop offered a forum for participants to address issues of ADs that arise at the planning, designing, and execution stages of clinical trials, and to hear the perspectives of influential members of the clinical trials community. The participants also set forth recommendations for guiding action to promote the appropriate use of ADs. These recommendations have since been presented, discussed, and vetted in a number of venues including the University of Pennsylvania Conference on Statistical Issues in Clinical Trials and the Society for Clinical Trials annual meeting. PURPOSE: To provide a brief overview of ADs, describe the rationale behind conducting the workshop, and summarize the main recommendations that were produced as a result of this workshop. CONCLUSIONS: There is a growing interest in the use of adaptive clinical trial designs. However, a number of logistical barriers need to be addressed in order to obtain the potential advantages of an AD. Currently, the pharmaceutical industry is well ahead of academic trialists with respect to addressing these barriers. Academic trialists will need to address important issues such as education, infrastructure, modifications to existing funding models, and the impact on Data and Safety Monitoring Boards (DSMB) in order to achieve the possible benefits of adaptive clinical trial designs.

The evolution and impact of testing baghouse filter performance.

In 1995, the US. Environmental Protection Agency (EPA) initiated the Environmental Technology Verification (ETV) program for the purpose of generating both independent and credible performance verification of innovative technologies and helping to accelerate acceptance of these products into the marketplace to further benefit the environment and protect public health. The EPA has approved a testing protocol under this program to verify the performance of commercially available filtration products for pulse-jet baghouses in removingfine particulate matter (aerodynamic diameter<2.5 microm; PM2.5). This verification testing protocol was later used as a basis for the development of the American Society for Testing and Materials (ASTM) Method D6830-02 and the International Organization for Standardization (ISO) Method 11057. The South Coast Air Quality Management District (SCAQMD) in California and the EPA s Office of Air Quality Planning and Standards (OAQPS) highly encourage the use of ETV/ASTM-verified filtration media. This paper highlights the evolution of the standard test methods, the EPA's and SCAQMD's regulatory activities, the benefits of using verified filtration media, and the importance of including the filter performance testing in future consideration of baghouse permitting, baghouse operation and maintenance (O&M) plans, quality assurance/quality control (QA/QC), and bag monitoring plans.

Single-blind, randomized controlled trial evaluating the treatment of facial seborrheic dermatitis with hydrocortisone 1% ointment compared with tacrolimus 0.1% ointment in adults.

BACKGROUND: Tacrolimus is a topical calcineurin inhibitor with immunomodulatory, anti-inflammatory, and fungicidal properties that may be beneficial in the treatment of facial seborrheic dermatitis. OBJECTIVES: We sought to compare the efficacy and safety of tacrolimus with standard corticosteroid treatment in adults with facial seborrheic dermatitis in a phase II, single-blind, randomized controlled trial. METHODS: Adult patients were enrolled in a 12-week study. Subjects were randomized to tacrolimus 0.1% ointment (n = 16) or hydrocortisone 1% ointment (n = 14) applied twice daily to symptomatic regions of the face. The primary efficacy measure was the severity of facial seborrhea at the end of treatment (day 84) as measured by the Seborrhea Area and Severity Index-Face. Secondary efficacy measures included physician and patient assessment of seborrhea, the frequency of medication application, and adverse events. RESULTS: The severity of facial seborrhea was similarly improved in both treatment groups (P = .86). Tacrolimus 0.1% ointment was used on significantly fewer days than 1% hydrocortisone ointment (mean missed doses per patient at first visit: 15.6 vs 7.6, P < .05; at last visit: 13.5 vs 7.7, P = .08). The majority of doses were missed because of lack of symptoms. The adverse event profile for both agents was similar; however, there was a numerically higher incidence of adverse events in the hydrocortisone group. LIMITATIONS: This was a small, open-label study. CONCLUSION: Tacrolimus 0.1% ointment required significantly fewer applications compared with hydrocortisone 1% ointment to achieve a comparable clinical response in adults with facial seborrheic dermatitis. Tacrolimus was generally well tolerated.

Psychopathology, trauma and delinquency: subtypes of aggression and their relevance for understanding young offenders.

OBJECTIVE: To examine the implications of an ontology of aggressive behavior which divides aggression into reactive, affective, defensive, impulsive (RADI) or "emotionally hot"; and planned, instrumental, predatory (PIP) or "emotionally cold." Recent epidemiological, criminological, clinical and neuroscience studies converge to support a connection between emotional and trauma related psychopathology and disturbances in the emotions, self-regulation and aggressive behavior which has important implications for diagnosis and treatment, especially for delinquent populations. METHOD: Selective review of preclinical and clinical studies in normal, clinical and delinquent populations. RESULTS: In delinquent populations we observe an increase in psychopathology, and especially trauma related psychopathology which impacts emotions and self-regulation in a manner that hotly emotionally charged acts of aggression become more likely. The identification of these disturbances can be supported by findings in cognitive neuroscience. These hot aggressive acts can be delineated from planned or emotionally cold aggression. CONCLUSION: Our findings support a typology of diagnostic labels for disruptive behaviors, such as conduct disorder and oppositional defiant disorder, as it appears that these acts of hot emotional aggression are a legitimate target for psychopharmacological and other trauma specific interventions. The identification of this subtype of disruptive behavior disorders leads to more specific clinical interventions which in turn promise to improve hitherto unimpressive treatment outcomes of delinquents and patients with disruptive behavior.

Role of oxidative stress in geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex.

Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor geldanamycin (GA) cytotoxicity has been attributed to the disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1 h, and cell death occurred at 6 h, inhibited by N-acetylcysteine (NAC) without preventing dissociation of proteins. At 24 h, NAC prevented cytotoxicity and Hsp90 complex disruption. However, MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring glutathione. In contrast, 24 h MEN treatment induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding protein expression, and proteasomal inhibition prevented MEN-, but not GA-induced degradation. In conclusion, whereas MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces Hsp90 complex dissociation and proteasome-independent protein degradation. These differences between MEN- and GA-induced cytotoxicity may allow more specific targeting of cancer cells.

Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial cells.

Shigella flexneri is a facultative intracellular organism that causes bacillary dysentery. The Shigella IpaB protein activates caspase 1 in macrophages, which eventually leads to apoptosis. In contrast, epithelial cells infected with Shigella undergo a stress response but do not die. Therefore, the objective of this study was to determine if Shigella has the ability to inhibit apoptosis in epithelial cells. A modified gentamicin protection assay was used to investigate if HeLa cells infected with S. flexneri are able to resist the induction of apoptosis following treatment with 4 microM of staurosporine. Nuclear staining and immunofluorescence revealed that infected cells remained healthy while uninfected cells appeared apoptotic. Only uninfected cells had detectable levels of activated caspase 3 upon immunofluorescence, and this was verified by Western blot analysis. Despite interfering with caspase 3 activation, Shigella-infected cells treated with staurosporine did have cytochrome c release and caspase 9 activation, indicating that Shigella protects epithelial cells from apoptosis by inhibiting caspase 3 activation. Analysis of S. flexneri mutants showed that invasion and a functional type III secretion system were required to block apoptosis. In addition, a mutant with a deletion in mxiE, which encodes a transcriptional activator for genes induced intracellularly, failed to inhibit apoptosis. Therefore, protection of epithelial cells from apoptosis by S. flexneri is regulated by one or more of the bacterial genes under the control of mxiE. We believe that S. flexneri, like other pathogens, inhibits apoptosis in epithelial cells but causes apoptosis in macrophages to ensure survival inside the host.

Unique features of a highly pathogenic Campylobacter jejuni strain.

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain.

1alpha,25-dihydroxyvitamin D3 and bryostatin-1 synergize to induce monocytic differentiation of NB4 acute promyelocytic leukemia cells by modulating cell cycle progression.

This study examines the role of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and the natural compound, bryostatin-1, on the monocytic differentiation of NB4 acute promyelocytic leukemia cells. We previously showed that 1,25(OH)(2)D(3) primes NB4 cells to mature along the monocyte/macrophage pathway in response to the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). This maturation response involves protein kinase C (PKC) signaling, activation of the transcription factor nuclear factor kappaB (NFkB), and intracellular calcium and calpain activity. The natural compound, bryostatin-1, exhibits some of the effects of TPA but lacks its tumor-promoting nature. 1,25(OH)(2)D(3) treatment followed by bryostatin-1 induces monocytic differentiation of NB4 cells, however,this effect is less pronounced than the combination of 1,25(OH)(2)D(3) and TPA. Maturation is accompanied by decreased proliferation, changes in cellular morphology, increased plastic adherence, and expression of the cell surface marker CD14. Changes in the cell cycle traverse occur before the morphological and biochemical changes associated with differentiation. Within 24 h of bryostatin-1 addition, NB4 cells begin arresting, predominantly in G(1) phase. Changes in the cell cycle traverse were accompanied by changes in the expression of several cell cycle regulatory proteins. Combination 1,25(OH)(2)D(3) and bryostatin-1 treatment, resulted in decreased expression of the cyclin-dependent kinases Cdk2, Cdk1, and Cdk4, of cyclins E and D3, and of the retinoblastoma binding protein (RBBP). Levels of the cyclin-dependent kinase inhibitors p21 and p27 as well as Cyclin D1 were undetectable in NB4 cell lysates, suggesting that they do not participate in the differentiation response or cell cycle control in this model.

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.