Translational Control Of Protein Synthesis In Heat-shocked Maize Seedlings

Translational control of protein synthesis in the plumules of heat-shocked maize seedlings was investigated. Maize plumules respond to heat shock by synthesizing nuclear-encoded hsp\u27s with M{dollar}\sb{lcub}\rm r{rcub}{dollar}\u27s of 108, 89, 84, 76, 73, and 17-29kDa. Synthesis of some, but not all, 25{dollar}\sp\circ{dollar}C (control) proteins is repressed in vivo. RNA blot and in vitro translation analyses of non-polyribosomal and polyribosomal RNP RNA suggest that repression ensues from either a decrease in mRNA levels or from the inefficient translation of control messages in heat-shocked cells (relative to heat shock mRNAs) due to changes in the rates of both initiation and elongation.;Hsp70 and hsp18 mRNAs (or heat shock-like mRNAs) are synthesized at low levels in nonstressed cells. During heat shock, the messages accumulate on polyribosomes within 5 to 10 minutes, are maximal within 1 to 2h and decline thereafter (ie. maize plumules acclimate). The recovery profile is similar. Dissociation of message from the polyribosomes under these conditions is not accompanied by a re-association of heat shock mRNAs with non-polyribosomal RNPs suggesting that (1) the messages released from the polyribosomes are degraded and (2) the mechanism(s) governing these responses is independent of the incubation temperature. Acclimation and recovery are not characterized by a complete return to the control state as low molecular weight heat shock mRNAs continue to associate with the polyribosomal and non-polyribosomal fractions.;Heat shock and control mRNAs are differentially distributed in the non-polyribosomal RNP of control cells. However, messages active in translation at 25{dollar}\sp\circ{dollar}C are primarily associated with cytoskeletally-associated polyribosomes. Heat shock results in a change in both the distribution of heat shock mRNAs on the ribosomes of polyribosomes and their non-polyribosomal to polyribosomal ratios. Heat shock mRNAs accumulate primarily in the free-cytoplasmic non-polyribosomal and polyribosomal fractions suggesting that translation and possibly transport of these messages during heat shock is largely independent of the cytoskeleton. The negligible changes in the equilibrium of membrane-derived non-polyribosomal fractions together with the accumulation of mRNAs destined for the endoplasmic reticulum in free-cytoplasmic non-polyribosomal RNP are consistent with this interpretation. Neither the distribution of 25{dollar}\sp\circ{dollar}C mRNAs on polyribosomes nor their equilibrium between non-polyribosomal and polyribosomal RNP\u27s is affected by the temperature shift.;The kinetics of association/dissociation of mRNAs with the non-polyribosomal and polyribosomal RNP indicate that heat shock messages associate with non-polyribosomal RNP prior to their integration into polyribosomes

Design of a Debridement Device Using Impinging Jets

Chronic wound care is a significant burden on the healthcare system, affecting an estimated three to six million Americans, manifesting as ulcers associated with restricted blood flow, diabetes mellitus, or pressure. Treatment is frequently unsuccessful, with only an estimated 25–50% of venous and diabetic ulcers closing after 20 weeks of treatment. Debridement, the removal of necrotic tissue and foreign materials from the wounds, is a crucial component in the chronic wound care. While there exist many debridement techniques, the search for new and more effective methods is ongoing. The existing methods of debridement include surgical, the industry gold standard, as well as the mechanical, autolytic, enzymatic, and hydrosurgery (VersaJet™). The VersaJet™ uses a single high-speed jet directed parallel to the wound surface to remove soft necrotic tissue. This paper presents the design of a debridement device that uses two narrow, high-speed impinging fluid jets to excise necrotic tissue. The handheld device can be used to remove strips of necrotic tissue of a predetermined width and depth and was tested on samples of simulated slough, the soft necrotic tissue, and eschar, the hard, scablike necrotic tissue. The preliminary tests indicate that the technique removes necrotic tissue quickly and with good control, suggesting that, with further development, the technique may provide a time-saving alternative to surgical debridement. Further testing, however, is required to ensure that the jets do not damage the surrounding healthy tissues and to quantitatively analyze the effectiveness of the technique relative to other debridement strategies

Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs

Control of Candida albicans Metabolism and Biofilm Formation by Pseudomonas aeruginosa Phenazines

Candidaalbicanshasdevelopmentalprogramsthatgoverntransitionsbetweenyeastandfilamentousmorphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm develop- ment were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fer- mentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specif- ically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fer- mentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may sug- gest new ways to limit fungal biofilms in the context of disease

Mitochondrial Activity and Cyr1 are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology

Analysis of Candida albicans Mutants Defective in the Cdk8 Module of Mediator Reveal Links between Metabolism and Biofilm Formation

Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module-Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development

Patient Centeredness in Electronic Communication: Evaluation of Patient-to-Health Care Team Secure Messaging

BACKGROUND: As information and communication technology is becoming more widely implemented across health care organizations, patient-provider email or asynchronous electronic secure messaging has the potential to support patient-centered communication. Within the medical home model of the Veterans Health Administration (VA), secure messaging is envisioned as a means to enhance access and strengthen the relationships between veterans and their health care team members. However, despite previous studies that have examined the content of electronic messages exchanged between patients and health care providers, less research has focused on the socioemotional aspects of the communication enacted through those messages. OBJECTIVE: Recognizing the potential of secure messaging to facilitate the goals of patient-centered care, the objectives of this analysis were to not only understand why patients and health care team members exchange secure messages but also to examine the socioemotional tone engendered in these messages. METHODS: We conducted a cross-sectional coding evaluation of a corpus of secure messages exchanged between patients and health care team members over 6 months at 8 VA facilities. We identified patients whose medical records showed secure messaging threads containing at least 2 messages and compiled a random sample of these threads. Drawing on previous literature regarding the analysis of asynchronous, patient-provider electronic communication, we developed a coding scheme comprising a series of a priori patient and health care team member codes. Three team members tested the scheme on a subset of the messages and then independently coded the sample of messaging threads. RESULTS: Of the 711 messages coded from the 384 messaging threads, 52.5% (373/711) were sent by patients and 47.5% (338/711) by health care team members. Patient and health care team member messages included logistical content (82.6%, 308/373 vs 89.1%, 301/338), were neutral in tone (70.2%, 262/373 vs 82.0%, 277/338), and respectful in nature (25.7%, 96/373 vs 33.4%, 113/338). Secure messages from health care team members sometimes appeared hurried (25.4%, 86/338) but also displayed friendliness or warmth (18.9%, 64/338) and reassurance or encouragement (18.6%, 63/338). Most patient messages involved either providing or seeking information; however, the majority of health care team member messages involved information provision in response to patient questions. CONCLUSIONS: This evaluation is an important step toward understanding the content and socioemotional tone that is part of the secure messaging exchanges between patients and health care team members. Our findings were encouraging; however, there are opportunities for improvement. As health care organizations seek to supplement traditional encounters with virtual care, they must reexamine their use of secure messaging, including the patient centeredness of the communication, and the potential for more proactive use by health care team members

Observational study to determine the utility of hospital administrative data to support case finding of English patients at higher risk of severe healthcare-related harm.

OBJECTIVES: To identify ways of using routine hospital data to improve the efficiency of retrospective reviews of case records for identifying avoidable severe harm DESIGN: Development and testing of thresholds and criteria for two indirect indicators of healthcare-related harm (long length of stay (LOS) and emergency readmission) to determine the yield of specified harms coded in Hospital Episode Statistics (HES). SETTING: Acute National Health Service hospitals in England. PARTICIPANTS: HES for acute myocardial infarction (AMI), bowel cancer surgery and hip replacement admissions from 2014 to 2015. INTERVENTIONS: Case-mix-adjusted linear regression models were used to determine expected LOS. Different thresholds were examined to determine the association with harm. Screening criteria for readmission included time to readmission, length of readmission and diagnoses in initial admission and readmission. The association with harm was examined for each criterion. RESULTS: The proportions of AMI cases with a harm code increased from 14% among all cases to 47% if a threshold of three times the expected LOS was used. For hip replacement the respective increase was from 10% to 51%. However as the number of patients at these higher thresholds was small, the overall proportion of harm identified is relatively small (15%, 19%, 9% and 8% among AMI, urgent bowel surgery, elective bowel surgery and hip replacement cohorts, respectively). Selection of the time to readmission had an effect on the yield of harms but this varied with condition. At least 50% of surgical patients had a harm code if readmitted within 7 days compared with 21% of patients with AMI. CONCLUSIONS: Our approach would select a substantial number of patients for case record review. Many of these cases would contain no evidence of healthcare-related harm. In practice, Trusts may choose how many reviews it is feasible to do in advance and then select random samples of cases that satisfy the screening criteria

Pseudomonas Aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus Aureus in a Model of Cystic Fibrosis Respiratory Infection

While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example, Pseudomonas aeruginosa and Staphylococcus aureus are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that P. aeruginosa and/or S. aureus adopts phenotypes that allow coexistence during infection. We compared competitive interactions of P. aeruginosa and S. aureus isolates from mono- or coinfected CF patients employing in vitro coculture models. P. aeruginosa isolates from monoinfected patients were more competitive toward S. aureus than P. aeruginosa isolates from coinfected patients. We also observed that the least competitive P. aeruginosa isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill S. aureus. Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureus activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids—each required for efficient killing of S. aureus. These studies demonstrate alginate overproduction may be an important factor driving P. aeruginosa coinfection with S. aureus