Hypericum perforatum to improve post-operative Pain Outcome after monosegmental Spinal microdiscectomy (HYPOS): a study protocol for a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Spinal disc herniation is a frequently occurring degenerative disease of the spine. Many patients undergoing surgery suffer from radicular pain, known as memory pain, beginning from the third post-operative day. This results in the prescription of high-dose opioid medications. In homeopathy, Hypericum perforatum is known as a remedy for unbearable, shooting or jabbing pain especially when neural damage is involved. Reduction of pain after application of H. perforatum has been observed in previous studies. This study is aimed to investigate whether homeopathic H. perforatum in a potentisation of C200 leads to the reduction of post-operative pain and a decrease of pain medication compared to placebo. METHODS/DESIGN: This is a monocentric, double-blind, randomised placebo-controlled trial conducted at the Department of Neurosurgery at the Community Hospital Herdecke, Germany. One hundred study participants are being recruited from inpatients undergoing elective, monosegmental, lumbar microdiscectomy surgery. Patients are randomly allocated to receive homeopathic treatment or placebo in addition to usual pain management after surgery. The primary clinical outcome is pain reduction after 3 days of inpatient care as measured by pain reduction of subjective pain on a 100-mm Visual Analogue Scale (VAS) at the third post-operative day. Statistical analysis will be carried out by means of a covariance model with adjustment for baseline values and patient expectation for all randomised patients. DISCUSSION: This study is the first trial of classical homeopathy that will evaluate the efficacy of homeopathic H. perforatum after monosegmental spinal microdiscectomy. We intend to clarify the potential of homoeopathic H. perforatum to reduce surgery-associated pain. TRIAL REGISTRATION: German Clinical Trials Register, ID: DRKS00007913 . Registered on 17 October 2014. EudraCT - Nr: 2013-001383-31. Data sets from the German Clinical Trials Register (DRKS, Deutsches Register Klinischer Studien) are updated every 4 weeks automatically to the International Clinical Trials Registry Platform of World Health Organisation: http://apps.who.int/trialsearch/ . Responsibilities Sponsor: Witten/Herdecke University Alfred-Herrhausen-Straße 50 58,448 Witten Deputy of the sponsor: Dr. Wolfgang Eglmeier (Head of Centre for Clinical Trials Witten/Herdecke) Alfred-Herrhausen-Straße 50 58,448 Witten E-mail: wolfgang.eglmeier@uni-wh.de Principal investigator: Prof. Dr. med. Wolfram Scharbrodt Community Hospital Herdecke Department for Neurosurgery Gerhard-Kienle-Weg 4 58,313 Herdecke w.scharbrodt@gemeinschaftskrankenhaus.de Project coordination: Christa Raak Faculty for Health (Department for Integrative and Anthroposophic Medicine) University Witten/Herdecke gGmbh Gerhard-Kienle-Weg 4 58,313 Herdecke christa.raak@uni-wh.de Project manager/data analysis/biometry: Prof. Dr. Thomas Ostermann Faculty for Health (Department for Psychology and Psychotherapy) University Witten/Herdecke gGmbh Alfred-Herrhausen-Straße 50 58,448 Witten thomas.ostermann@uni-wh.de.

Dom34 Links Translation to Protein O-mannosylation.

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

6S RNA - an old issue became blue-green.

6S RNA from Escherichia coli acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with E. coli. To test for potential structural and functional conservation, we selected four predicted cyanobacterial 6S RNAs (Synechocystis, Synechococcus, Prochlorococcus and Nostoc), which we compared with their E. coli counterpart. Temperature-gradient gel electrophoresis revealed similar thermodynamic transition profiles for all 6S RNAs, indicating basically similar secondary structures. Subtle differences in melting behaviour of the different RNAs point to minor structural variations possibly linked to differences in optimal growth temperature. Secondary structural analysis of three cyanobacterial 6S RNAs employing limited enzymic hydrolysis and in-line probing supported the predicted high degree of secondary structure conservation. Testing for functional homology we found that all cyanobacterial 6S RNAs were active in binding E. coli RNA polymerase and transcriptional inhibition, and had the ability to act as template for transcription of product RNAs (pRNAs). Deletion of the 6S RNA gene in Synechocystis did not significantly affect cell growth in liquid media but reduced fitness during growth on solid agar. While our study shows that basic 6S RNA functions are conserved in species as distantly related as E. coli and cyanobacteria, we also noted a subtle degree of divergence, which might reflect fundamental differences in transcriptional regulation and lifestyle, thus providing the first evidence for a possible physiological role in cyanobacteria.

E. coli 6S RNA: a universal transcriptional regulator within the centre of growth adaptation.

Bacterial 6S RNA has been shown to bind with high affinity to σ(70)-containing RNA polymerase, suppressing σ(70)-dependent transcription during stationary phase, when 6S RNA concentrations are highest. We recently reported a genome-wide transcriptional comparison of wild-type and 6S RNA deficient E. coli strains. Contrary to the expected σ(70)- and stationary phase-specific regulatory effect of 6S RNA it turned out that mRNA levels derived from many alternative sigma factors, including σ(38) or σ(32), were affected during exponential and stationary growth. Among the most noticeably down-regulated genes at stationary growth are ribosomal proteins and factors involved in translation. In addition, a striking number of mRNA levels coding for enzymes involved in the purine metabolism, for transporters and stress regulators are altered both during log- and stationary phase. During the study we discovered a link between 6S RNA and the general stress alarmone ppGpp, which has a higher basal level in cells deficient in 6S RNA. This finding points to a functional interrelation of 6S RNA and the global network of stress and growth adaptation.

Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery.

BACKGROUND: 6S RNA from E. coli is known to bind to RNA polymerase interfering with transcription initiation. Because 6S RNA concentrations are maximal at stationary phase and binding occurs preferentially to the holoenzyme associated with sigma(70) (Esigma(70)) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for sigma(70)-dependent promoters characterized by a weak -35 recognition motif or extended -10 promoters. There are many exceptions to this precept, showing that other types of promoters, including stationary phase-specific (sigma(38)-dependent) promoters are inhibited. RESULTS: To solve this apparent ambiguity and to better understand the role of 6S RNA in stationary phase transition we have performed a genome-wide transcriptional analysis of wild-type and 6S RNA deficient cells growing to mid-log or early stationary phase. We found 245 genes at the exponential growth phase and 273 genes at the early stationary phase to be > or = 1.5-fold differentially expressed. Up- and down-regulated genes include many transcriptional regulators, stress-related proteins, transporters and several enzymes involved in purine metabolism. As the most striking result during stationary phase, however, we obtained in the 6S RNA deficient strain a concerted expression reduction of genes constituting the translational apparatus. In accordance, primer extension analysis showed that transcription of ribosomal RNAs, representing the key molecules for ribosome biogenesis, is also significantly reduced under the same conditions. Consistent with this finding biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S RNA is apparently compensated by an increase of the basal ppGpp concentration, known to affect growth adaptation and ribosome biogenesis. CONCLUSIONS: The analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to sigma(70)-dependent promoters. Moreover, the results indicate that 6S RNA is embedded in stationary phase adaptation, which is governed by the capacity of the translational machinery.

Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS.

Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.