Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

<p>Abstract</p> <p>Background</p> <p>Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium <it>Pseudomonas syringae </it>pv. <it>syringae </it>B728a with its host plant <it>Phaseolus vulgaris </it>(bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames <it>sylA-sylE </it>allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.</p> <p>Results</p> <p>We have cloned a 22 kb genomic fragment containing the <it>sylA-sylE </it>gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into <it>Pseudomonas putida </it>and <it>P. syringae </it>pv. <it>syringae </it>SM was sufficient to direct the biosynthesis of <it>bona fide </it>syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH¹³CO₃revealed preferential ¹³C-labeling at the ureido carbonyl position.</p> <p>Conclusion</p> <p>The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the <it>sylC </it>gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.</p

Correlation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read-out

Biofilm-related infections occur quite frequently in hospital settings and require rapid diagnostic identification as they are recalcitrant to antibiotic therapy and make special treatment necessary. One of the standard microbiological in vitro tests is the crystal violet test. It indirectly determines the amount of biofilm by measuring the optical density (OD) of the crystal violet-stained biofilm matrix and cells. However, this test is quite time-consuming, as it requires bacterial cultivation up to several days. In this study, we correlate fast Raman spectroscopic read-out of clinical Staphylococcus aureus isolates from 47 patients with different disease background with their biofilm-forming characteristics. Included were low (OD  20) biofilm performers as determined by the crystal violet test. Raman spectroscopic analysis of the bacteria revealed most spectral differences between high and low biofilm performers in the fingerprint region between 750 and 1150 cm−1. Using partial least square regression (PLSR) analysis on the Raman spectra involving the three categories of biofilm formation, it was possible to obtain a slight linear correlation of the Raman spectra with the biofilm OD values. The PLSR loading coefficient highlighted spectral differences between high and low biofilm performers for Raman bands that represent nucleic acids, carbohydrates, and proteins. Our results point to a possible application of Raman spectroscopy as a fast prediction tool for biofilm formation of bacterial strains directly after isolation from the infected patient. This could help clinicians make timely and adapted therapeutic decision in future

Probing shock geometry via the charge to mass ratio dependence of heavy ion spectra from multiple spacecraft observations of the 2013 November 4 event

In large Solar Energetic Particle (SEP) events, ions can be accelerated at coronal mass ejection (CME)-driven shocks to very high energies. The spectra of heavy ions in many large SEP events show features such as roll-overs or spectral breaks. In some events when the spectra are plotted in terms of energy/nucleon, they can be shifted relative to each other to make the spectral breaks align. The amount of shift is charge to mass ratio (Q/A) dependent and varies from event to event. This can be understood if the spectra of heavy ions are organized by the diffusion coefficients (Cohen et al. 2005). In the work of Li et al. (2009), the Q/A dependence of the scaling is related to shock geometry when the CME-driven shock is close to the Sun. For events where multiple in-situ spacecraft observations exist, one may expect that different spacecraft are connected to different portions of the CME-driven shock that have different shock geometries, therefore yielding different Q/A dependence. In this work, we examine one SEP event which occurred on 2013 November 4. We study the Q/A dependence of the energy scaling for heavy ion spectra using helium, oxygen and iron ions. Observations from STEREO-A, STEREO-B and ACE are examined. We find that the scalings are different for different spacecraft. We suggest that this is because ACE, STEREO-A and STEREO-B are connected to different parts of the shock that have different shock geometries. Our analysis indicates that studying the Q/A scaling of in-situ particle spectra can serve as a powerful tool to remotely examine the shock geometry for large SEP events

Children of Mentally III Parents at Risk Evaluation (COMPARE): Design and Methods of a Randomized Controlled Multicenter Study—Part I

Objectives: Mental disorders are frequent, associated with disability-adjusted life years, societal, and economic costs. Children of parents with a mental illness (COPMI) are at an increased risk to develop disorders themselves. The transgenerational transmission of mental disorders has been conceptualized in a model that takes parental and family factors, the social environment (i.e., school, work, and social support), parent-child-interaction and possible child outcomes into account. The goal of the “Children of Mentally Ill Parents At Risk Evaluation” (COMPARE) study will thus be twofold: (1) to establish the efficacy and cost-effectiveness of a high-quality randomized controlled trial (RCT) with the aim of interrupting the intergenerational transmission of mental disorders in COPMI, (2) to test the components of the trans-generational transmission model of mental disorders. Methods: To implement a randomized controlled trial (RCT: comparison of parental cognitive behavioral therapy/CBT with CBT + Positive Parenting Program) that is flanked by four add-on projects that apply behavioral, psychophysiological, and neuro-imaging methods to examine potential moderators and mediators of risk transmission (projects COMPARE-emotion/-interaction/-work/-school). COMPARE-emotion targets emotion processing and regulation and its impact on the transgenerational disorder transmission; COMPARE-interaction focuses especially on the impact of maternal comorbid diagnoses of depression and anxiety disorders and will concentrate on different pathways of the impact of maternal disorders on socio-emotional and cognitive infant development, such as parent-infant interaction and the infant's stress regulation skills. COMPARE-work analyzes the transmission of strains a person experiences in one area of life to another (i.e., from family to work; spill-over), and how stress and strain are transmitted between individuals (i.e., from parent to child; cross-over). COMPARE-school focuses on the psychosocial adjustment, school performance, and subjective well-being in COPMI compared to an adequate control group of healthy children. Results: This study protocol reports on the interdisciplinary approach of COMPARE testing the model of the transgenerational transmission of mental disorders. Conclusion: The combination of applied basic with clinical research will facilitate the examination of specific risk transmission mechanisms, promotion, dissemination and implementation of results into a highly important but largely neglected field. Clinical Trial Registration: DRKS-ID: DRKS00013516 (German Clinical Trials Register, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013516)

Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence

Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD, Caarv1C3S, and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution

COMPARE Family (Children of Mentally Ill Parents at Risk Evaluation): A Study Protocol for a Preventive Intervention for Children of Mentally Ill Parents (Triple P, Evidence-Based Program That Enhances Parentings Skills, in Addition to Gold-Standard CBT With the Mentally Ill Parent) in a Multicenter RCT—Part II

Background: Mental health problems are highly frequent, as well as being associated with enormous societal and economic costs and significant disability-adjusted life years. Children of parents with a mental illness (COPMI) are at a tremendously increased risk to develop disorders themselves. According to the literature, parental mental disorders launch a wave of risk factors that in turn predict the emergence of psychological problems in the offspring, and effective treatment of the parental disorder has been associated with reduced child psychopathology (launch and grow assumption). Furthermore, studies focusing on parent-child interaction demonstrate generally poorer parenting skills in parents with mental disorders, and the enhancement of such skills has been a significant mediator in improving child outcomes (parenting assumption).Objective: To implement a preventive intervention for COPMI with the aim of interrupting the transmission of mental disorders in children of a parent with mental disorders. An RCT will compare state-of-the-art cognitive behavioral therapy (CBT) for a parent with mental disorders to CBT plus the Positive Parenting Program (Triple-P), a well-established and evidence-based program that enhances parenting skills.Methods: A total of 634 patients seeking treatment in 8 outpatient clinics in Germany and their children will be included between January 2018 and April 2021 in the study. We use (clinical) interviews and self- as well as other-report questionnaires to assess the families at four main measurement points [T1: beginning of waiting period for psychotherapy treatment (duration of waiting period depends on usual waiting period in the study center: multiple baselines), T2: begin of parental psychotherapy, T3: post-assessment, T4: 6 months follow-up]. The total observation period will be 39 months. The patients will be randomly assigned to either the control condition (25 to 45 CBT sessions) or the experimental condition (25 to 45 CBT sessions + 10 Triple-P sessions). For evaluating the treatment process, the patients and clinicians will also be assessed after each treatment session. Furthermore, there will be a continuous assessment and report of adverse events during treatment.Discussion: This trial will be the first ever to address the launch and grow as well as the parenting assumption in one study and to establish effects of the two different interventions on children's health. Our study will also likely be the first one to provide data on the comparative cost-effectiveness and will therefore provide essential information relevant for the potential implementation of such programs. The structure of the RCT will allow us to establish effects of the parental disorder(s) with/without comorbidities on children's health, to test assumptions of the trans-generational transmission model of mental disorders and bi-directional influences of different treatments on the model and to analyze specific transmission mechanisms. A deeper understanding of risk mechanisms will reveal specific transmission profiles that will result in the early detection of and effective reduction in risk factors and thus improve the health of the children at risk.Ethics: The study is carried out according to the Good Clinical Practice (GCP) guidelines, the Declaration of Helsinki and its later supplements and local legal requirements. The lead ethics committee at the department of psychology at Philipps-University Marburg approved the study procedure and all study documents. A positive ethics committee vote is required at a study site, before the inclusion of a first patient at the respective site.Dissemination: Via peer-reviewed publications in scientific journals, the results of this study will be made available to the scientific community. Using PsychData all primary data will be made available for re- and meta-analyses. Politicians, public health services, and stakeholders will be informed throughout the study and beyond, thus, improving public policy and health care decisions concerning preventive interventions and treatments for COPMI.Trial Registration: DRKS-ID: DRKS00013516 (German Clinical Trials Register, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&amp;TRIAL_ID=DRKS00013516

Suppression of endothelial miR-22 mediates non-small cell lung cancer cell-induced angiogenesis

MicroRNAs (miRNAs) expressed in endothelial cells (ECs) are powerful regulators of angiogenesis, which is essential for tumor growth and metastasis. Here, we demonstrated that miR-22 is preferentially and highly expressed in ECs, while its endothelial level is significantly downregulated in human non-small cell lung cancer (NSCLC) tissues when compared to matched nontumor lung tissues. This reduction of endothelial miR-22 is possibly induced by NSCLC cell-secreted interleukin-1b and subsequently activated transcription factor nuclear factor-kB. Endothelial miR-22 functions as a potent angiogenesis inhibitor that inhibits all of the key angiogenic activities of ECs and consequently NSCLC growth through directly targeting sirtuin 1 and fibroblast growth factor receptor 1 in ECs, leading to inactivation of AKT/mammalian target of rapamycin signaling. These findings provide insight into the molecular mechanisms of NSCLC angiogenesis and indicate that endothelial miR-22 represents a potential target for the future antiangiogenic treatment of NSCLC