Phase 1 Open-Label Dose Escalation Trial for the Development of a Human Bacillus Calmette-Guérin Challenge Model for Assessment of Tuberculosis Immunity In Vivo.

BACKGROUND: A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development. METHODS: In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture. RESULTS: Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males. CONCLUSIONS: The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process. CLINICAL TRIALS REGISTRATION: NCT01868464 (ClinicalTrials.gov).

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13.

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment.

Potent, specific MEPicides for treatment of zoonotic staphylococci.

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

From graphene to graphene oxide: the importance of extended topological defects.

Graphene oxide (GO) represents a complex family of materials related to graphene: easy to produce in large quantities, easy to process, and convenient to use as a basis for further functionalization, with the potential for wide-ranging applications such as in nanocomposites, electronic inks, biosensors and more. Despite their importance, the key structural traits of GO, and the impact of these traits on properties, are still poorly understood due to the inherently berthollide character of GO which complicates the establishment of clear structure/property relationships. Widely accepted structural models of GO frequently neglect the presence of extended topological defects, structural changes to the graphene basal plane that are not removed by reduction methods. Here, a combination of experimental approaches and molecular simulations demonstrate that extended topological defects are a common feature across GO and that the presence of these defects strongly influences the properties of GO. We show that these extended topological defects are produced following even controlled 'gentle' functionalization by atomic oxygen and are comparable to those obtained by a conventional modified Hummers' method. The presence of the extended topological defects is shown to play an important role in the retention of oxygen functional groups after reduction. As an exemplar of their effect on the physical properties, we show that the GO sheets display a dramatic decrease in strength and stiffness relative to graphene and, due to the presence of extended structural defects, no improvement is seen in the mechanical properties after reduction. These findings indicate the importance of extended topological defects to the structure and properties of functionalized graphene, which merits their inclusion as a key trait in simple structural models of GO.

Alterations in cellular expression in EBV infected epithelial cell lines and tumors.

The Epstein Barr virus (EBV) is linked to the development of two major epithelial malignancies, gastric carcinoma and nasopharyngeal carcinoma. This study evaluates the effects of EBV on cellular expression in a gastric epithelial cell line infected with or without EBV and a nasopharyngeal carcinoma cell line containing EBV. The cells were grown in vitro and as tumors in vivo. The effects on cellular expression were determined using both 2D DIGE proteomics and high throughput RNA sequencing. The data identify multiple pathways that were uniquely activated in vitro. RNA sequences mapping to the mouse genome were identified in both the EBV positive and negative tumor samples in vivo, although, differences between the EBV positive and negative cells were not apparent. However, the tumors appeared to be grossly distinct. The majority of the identified canonical pathways based on two fold changes in expression had decreased activity within the tumors in vivo. Identification of the predicted upstream regulating factors revealed that in vitro the regulating factors were primarily protein transcriptional regulators. In contrast, in vivo the predicted regulators were frequently noncoding RNAs. Hierarchical clustering distinguished the cell lines and tumors, the EBV positive tumors from the EBV negative tumors, and the NPC tumors from the gastric tumors and cell lines. The delineating genes were changed greater than 4 fold and were frequently regulated by protein transcription factors. These data suggest that EBV distinctly affects cellular expression in gastric tumors and NPC and that growth in vivo requires activation of fewer cellular signaling pathways. It is likely that the broad changes in cellular expression that occur at low levels are controlled by regulatory viral and cellular RNAs while major changes are affected by induced protein regulators.

Improving management of fever in neutropenic children with cancer across multiple sites.

OBJECTIVE: To evaluate the effectiveness of a clinical pathway in achieving antibiotic administration in less than 60 minutes for children with cancer, presenting with fever and neutropenia. Secondary objectives were to determine association between time to antibiotics (TTA) and other variables including fever duration, location of care and intravenous access types. METHODS: Following introduction of the clinical pathway, we collected prospective data about management of all cases that did and did not use the pathway across multiple sites over 16 months. A follow-up audit was conducted after 12 months. RESULTS: We evaluated a total of 453 presentations. Use of the clinical pathway was significantly associated with achieving TTA in less than 60 minutes (RR 0.69, 95% CI 0.56-0.85, p = <0.001). Despite varying use of the pathway over time, the median time to antibiotics was achieved in both the initial study period (57 minutes) and sustained at follow-up (60 minutes). TTA was also associated with types of intravenous access device and location of care and with length of stay. We did not find any association between TTA and any other variables. CONCLUSION: Clinical pathways improve fever management in this patient cohort. Ongoing education and auditing to identify factors which impact processes of care are necessary.

Normal saline (0.9% sodium chloride) versus heparin intermittent flushing for the prevention of occlusion in long-term central venous catheters in infants and children.

BACKGROUND: Guidelines and clinical practice for the prevention of complications associated with central venous catheters (CVC) around the world vary greatly. Most institutions recommend the use of heparin to prevent occlusion; there is debate, however, regarding the need for heparin and evidence to suggest normal saline (0.9% sodium chloride) may be as effective. The use of heparin is not without risk, may be unnecessary and is also associated with increased cost. This is an update of the review published in 2015. OBJECTIVES: To assess the clinical effects (benefits and harms) of intermittent flushing of normal saline versus heparin to prevent occlusion in long-term central venous catheters in infants and children. SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Cochrane Vascular Specialised Register, CENTRAL, MEDLINE, Embase and CINAHL databases; World Health Organization International Clinical Trials Registry Platform and ClinicalTrials.gov trials register to 9 April 2019. We also undertook reference checking, citation searching and contact with study authors to identify additional studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared the efficacy of intermittent flushing with normal saline versus heparin to prevent occlusion of long-term CVCs in infants and children aged up to 18 years of age. We excluded temporary CVCs and peripherally inserted central catheters (PICC). DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial inclusion criteria, trial quality and extracted data. We assessed study quality with the Cochrane 'Risk of bias' tool. For dichotomous outcomes, we calculated the rate ratio (RR) and corresponding 95% confidence interval (CI). We pooled data using a random-effects model; and we used GRADE to assess the overall certainty of the evidence supporting the outcomes assessed in this review. MAIN RESULTS: We identified one new study for this update, bringing the total number of included studies to four (255 participants). The four trials directly compared the use of normal saline and heparin; the studies all used different protocols for the intervention and control arms, however, and all used different concentrations of heparin. Different frequencies of flushes were also reported between studies. In addition, not all studies reported on all outcomes. The certainty of the evidence ranged from moderate to very low because there was no blinding; heterogeneity and inconsistency between studies was high; and the CIs were wide. CVC occlusion was assessed in all four trials. We were able to pool the results of two trials for the outcomes of CVC occlusion and CVC-associated blood stream infection. The estimated RR for CVC occlusion per 1000 catheter days between the normal saline and heparin groups was 0.75 (95% CI 0.10 to 5.51; 2 studies, 229 participants; very low certainty evidence). The estimated RR for CVC-associated blood stream infection was 1.48 (95% CI 0.24 to 9.37; 2 studies, 231 participants; low-certainty evidence). The duration of catheter placement was reported to be similar for the two study arms in one study (203 participants; moderate-certainty evidence), and not reported in the remaining studies. AUTHORS' CONCLUSIONS: The review found that there was not enough evidence to determine the effects of intermittent flushing with normal saline versus heparin to prevent occlusion in long-term central venous catheters in infants and children. It remains unclear whether heparin is necessary to prevent occlusion, CVC-associated blood stream infection or effects duration of catheter placement. Lack of agreement between institutions around the world regarding the appropriate care and maintenance of these devices remains.

Prevention of occlusion of cEnTral lInes for children with cancer: An implementation study.

AIM: Central venous access devices (CVADs) are vital medical devices to support the treatment of paediatric cancer; however, device occlusion is common, which disrupts treatment. This study aimed to improve the identification and management of CVAD occlusions in children with cancer, as well as to identify the demographic, clinical and device characteristics associated with increased risk for CVAD occlusion. METHODS: A pre-post-implementation study was conducted at a metropolitan paediatric oncology facility in Australia, using the Theoretical Domains Framework. Patients with a CVAD for anti-cancer therapy were prospectively followed for occlusive events pre- and post- the implementation of clinical resources to support the identification and management of CVAD occlusive events. CVAD occlusion and management data were collected and compared pre- and post-implementation. Risk factors for CVAD occlusion were described by mixed-effects Poisson regression and incident rate ratios (IRR). RESULTS: A total of 133 CVADs were inserted into 131 patients for a total of 6784 catheter days. The incidence of CVAD-related occlusion pre-implementation was 59.7 (95% confidence interval (CI) 51.4-69.0, per 1000 catheter days); compared to 31.6 (95% CI 26.4-37.6); P < 0.01) post-implementation of clinical resources. In multivariate models, other than post-implementation phases (IRR 0.51 (95% CI 0.32-0.81)), only neutropaenia significantly increased the risk of CVAD occlusion (IRR 2.14 (95% CI 1.15-3.97)). CONCLUSION: CVAD occlusions in paediatric oncology are common. The development and implementation of CVAD occlusion resources to guide the identification and management of occlusive episodes led to a significant decrease in occlusive events.

Calculating the Magnetic Anisotropy of Rare-Earth-Transition-Metal Ferrimagnets.

Magnetocrystalline anisotropy, the microscopic origin of permanent magnetism, is often explained in terms of ferromagnets. However, the best performing permanent magnets based on rare earths and transition metals (RE-TM) are in fact ferrimagnets, consisting of a number of magnetic sublattices. Here we show how a naive calculation of the magnetocrystalline anisotropy of the classic RE-TM ferrimagnet GdCo_{5} gives numbers that are too large at 0 K and exhibit the wrong temperature dependence. We solve this problem by introducing a first-principles approach to calculate temperature-dependent magnetization versus field (FPMVB) curves, mirroring the experiments actually used to determine the anisotropy. We pair our calculations with measurements on a recently grown single crystal of GdCo_{5}, and find excellent agreement. The FPMVB approach demonstrates a new level of sophistication in the use of first-principles calculations to understand RE-TM magnets.

Laterally Transferred Gene Recruited as a Venom in Parasitoid Wasps.

Parasitoid wasps use venom to manipulate the immunity and metabolism of their host insects in a variety of ways to provide resources for their offspring. Yet, how genes are recruited and evolve to perform venom functions remain open questions. A recently recognized source of eukaryotic genome innovation is lateral gene transfer (LGT). Glycoside hydrolase family 19 (GH19) chitinases are widespread in bacteria, microsporidia, and plants where they are used in nutrient acquisition or defense, but have previously not been known in metazoans. In this study, a GH19 chitinase LGT is described from the unicellular microsporidia/Rozella clade into parasitoid wasps of the superfamily Chalcidoidea, where it has become recruited as a venom protein. The GH19 chitinase is present in 15 species of chalcidoid wasps representing four families, and phylogenetic analysis indicates that it was laterally transferred near or before the origin of Chalcidoidea (∼95 Ma). The GH19 chitinase gene is highly expressed in the venom gland of at least seven species, indicating a role in the complex host manipulations performed by parasitoid wasp venom. RNAi knockdown in the model parasitoid Nasonia vitripennis reveals that-following envenomation-the GH19 chitinase induces fly hosts to upregulate genes involved in an immune response to fungi. A second, independent LGT of GH19 chitinase from microsporidia into mosquitoes was also found, also supported by phylogenetic reconstructions. Besides these two LGT events, GH19 chitinase is not found in any other sequenced animal genome, or in any fungi outside the microsporidia/Rozella clade.

A Novel Fluorescence Resonance Energy Transfer-Based Screen in High-Throughput Format To Identify Inhibitors of Malarial and Human Glucose Transporters.

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC50) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening.

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region.

UNLABELLED: In Epstein-Barr virus-infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs (miRNAs), and the spliced and polyadenylated exons form nuclear non-protein-coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells; however, myc activation was confirmed through use of a myc-responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full-length cDNA clone of one of the BART isoforms was obtained and expressed in the Epstein-Barr virus (EBV)-negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription, as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells, where the BARTs are exclusively nuclear and do not encode proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system. IMPORTANCE: Infection with Epstein-Barr virus (EBV) is nearly ubiquitous in the human population; however, it does contribute to the formation of multiple types of cancer. In immunocompromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV-positive gastric carcinoma does not require immune suppression, and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein-coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first report to show that the highly expressed polyadenylated BamHI A rightward transcripts (BART) viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

Modulation of B-cell exosome proteins by gamma herpesvirus infection.

The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.

Heparin versus 0.9% sodium chloride intermittent flushing for the prevention of occlusion in long term central venous catheters in infants and children.

BACKGROUND: Guidelines and clinical practice for the prevention of complications associated with central venous catheters (CVC) around the world vary greatly. Most institutions recommend the use of heparin to prevent occlusion, however there is debate regarding the need for heparin and evidence to suggest 0.9% sodium chloride (normal saline) may be as effective. The use of heparin is not without risk, may be unnecessary and is also associated with increased cost. OBJECTIVES: To assess the clinical effects (benefits and harms) of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. SEARCH METHODS: The Cochrane Vascular Trials Search Co-ordinator searched the Specialised Register (last searched April 2015) and the Cochrane Register of Studies (Issue 3, 2015). We also searched the reference lists of retrieved trials. SELECTION CRITERIA: Randomised controlled trials that compared the efficacy of normal saline with heparin to prevent occlusion of long term CVCs in infants and children aged up to 18 years of age were included. We excluded temporary CVCs and peripherally inserted central catheters (PICC). DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial inclusion criteria, trial quality and extracted data. Rate ratios were calculated for two outcome measures - occlusion of the CVC and central line-associated blood stream infection. Other outcome measures included duration of catheter placement, inability to withdraw blood from the catheter, use of urokinase or recombinant tissue plasminogen, incidence of removal or re-insertion of the catheter, or both, and other CVC-related complications such as dislocation of CVCs, other CVC site infections and thrombosis. MAIN RESULTS: Three trials with a total of 245 participants were included in this review. The three trials directly compared the use of normal saline and heparin, however, between studies, all used different protocols for the standard and experimental arms with different concentrations of heparin and different frequency of flushes reported. In addition, not all studies reported on all outcomes. The quality of the evidence ranged from low to very low because there was no blinding, heterogeneity and inconsistency between studies was high and the confidence intervals were wide. CVC occlusion was assessed in all three trials (243 participants). We were able to pool the results of two trials for the outcomes of CVC occlusion and CVC-associated blood stream infection. The estimated rate ratio for CVC occlusion per 1000 catheter days between the normal saline and heparin group was 0.75 (95% CI 0.10 to 5.51, two studies, 229 participants, very low quality evidence). The estimated rate ratio for CVC-associated blood stream infection was 1.48 (95% CI 0.24 to 9.37, two studies, 231 participants; low quality evidence). The duration of catheter placement was reported to be similar between the two study arms, in one study (203 participants). AUTHORS' CONCLUSIONS: The review found that there was not enough evidence to determine the effects of intermittent flushing of heparin versus normal saline to prevent occlusion in long term central venous catheters in infants and children. Ultimately, if this evidence were available, the development of evidenced-based clinical practice guidelines and consistency of practice would be facilitated.

Laser-vibrometric ultrasonic characterization of resonant modes and quality factors of Ge membranes.

The vibrations of a single-crystal germanium (Ge) membrane are studied in air and vacuum using laser vibrometry, in order to determine mechanical properties such as Q-factors, tensile stress, anisotropy, and robustness to shock. Resonance modes up to 3:2 are identified, giving a residual stress measurement of 0.22 GPa, consistent with the value obtained from x-ray relaxation studies. The membrane is found to be isotropic, with Q-factors ranging from around 40 at atmospheric pressure to over 3200 at [Formula: see text] mbar, significantly lower than those found in polycrystalline Ge micromechanical devices. The robustness to shock is explained through the high resonance mode frequencies and the dissipation mechanism into the substrate, which is a direct consequence of having a high quality film with low residual tensile stress, giving the potential for such films to be used in optoelectronic devices.

Manage Your Life Online (MYLO): a pilot trial of a conversational computer-based intervention for problem solving in a student sample.

BACKGROUND: Computerized self-help that has an interactive, conversational format holds several advantages, such as flexibility across presenting problems and ease of use. We designed a new program called MYLO that utilizes the principles of METHOD of Levels (MOL) therapy--based upon Perceptual Control Theory (PCT). AIMS: We tested the efficacy of MYLO, tested whether the psychological change mechanisms described by PCT mediated its efficacy, and evaluated effects of client expectancy. METHOD: Forty-eight student participants were randomly assigned to MYLO or a comparison program ELIZA. Participants discussed a problem they were currently experiencing with their assigned program and completed measures of distress, resolution and expectancy preintervention, postintervention and at 2-week follow-up. RESULTS: MYLO and ELIZA were associated with reductions in distress, depression, anxiety and stress. MYLO was considered more helpful and led to greater problem resolution. The psychological change processes predicted higher ratings of MYLO's helpfulness and reductions in distress. Positive expectancies towards computer-based problem solving correlated with MYLO's perceived helpfulness and greater problem resolution, and this was partly mediated by the psychological change processes identified. CONCLUSIONS: The findings provide provisional support for the acceptability of the MYLO program in a non-clinical sample although its efficacy as an innovative computer-based aid to problem solving remains unclear. Nevertheless, the findings provide tentative early support for the mechanisms of psychological change identified within PCT and highlight the importance of client expectations on predicting engagement in computer-based self-help.

Problem-solving processes for central venous catheter occlusion within pediatric cancer care: A qualitative study.

PURPOSE: Central venous access devices play a crucial role in healthcare settings. However, there is concern regarding the high incidence of blockages occurring before the completion of treatments and existing guidelines for occlusion management are not consistently followed. To explore the decision-making and problem-solving process of occlusion management and identify enablers and barriers to implementing evidence for occlusion management in pediatric cancer care. METHODS: A qualitative design with individual semi-structured interviews. Participants were selected by purposeful sampling from a tertiary-referral pediatric facility, and semi-structured interviews were conducted. RESULTS: A total of 13 clinicians and 5 parents were interviewed. The thematic analysis revealed four main decision-making/problem-solving themes: 1) clinical reasoning and judgement for central venous access devices occlusion, 2) capability in central venous access devices occlusion management, 3) colleague collaboration in the escalation process and 4) lack of adequate support to manage the occlusion. This study identified positive and negative influences on the problem-solving process, including clinicians' psychological capabilities, social and physical resources, and beliefs about consequences. CONCLUSION: This study found that clinicians in pediatric cancer care were able to manage central venous access device occlusions using clinical reasoning and judgment skills, which may conflict with evidence-based practices. The study confirmed the importance of a team approach and prior experience in managing central venous access devices in pediatric oncology settings and identified potential conflicts between clinician decisions based on the patient's current and anticipated conditions and implementation of evidence-based practice. Improving documentation and providing visual aids could benefit clinicians' problem-solving processes.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Nicotinic acid modulates Legionella pneumophila gene expression and induces virulence traits.

In response to environmental fluctuations or stresses, bacteria can activate transcriptional and phenotypic programs to coordinate an adaptive response. The intracellular pathogen Legionella pneumophila converts from a noninfectious replicative form to an infectious transmissive form when the bacterium encounters alterations in either amino acid concentrations or fatty acid biosynthesis. Here, we report that L. pneumophila differentiation is also triggered by nicotinic acid, a precursor of the central metabolite NAD(+). In particular, when replicative L. pneumophila are treated with 5 mM nicotinic acid, the bacteria induce numerous transmissive-phase phenotypes, including motility, cytotoxicity toward macrophages, sodium sensitivity, and lysosome avoidance. Transcriptional profile analysis determined that nicotinic acid induces the expression of a panel of genes characteristic of transmissive-phase L. pneumophila. Moreover, an additional 213 genes specific to nicotinic acid treatment were altered. Although nearly 25% of these genes lack an assigned function, the gene most highly induced by nicotinic acid treatment encodes a putative major facilitator superfamily transporter, Lpg0273. Indeed, lpg0273 protects L. pneumophila from toxic concentrations of nicotinic acid as judged by analyzing the growth of the corresponding mutant. The broad utility of the nicotinic acid pathway to couple central metabolism and cell fate is underscored by this small metabolite's modulation of gene expression by diverse microbes, including Candida glabrata, Bordetella pertussis, Escherichia coli, and L. pneumophila.

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model.

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.