Fluorescence-based monitoring of ribosome assembly landscapes

Background Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. Results We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution. Conclusion This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents

Rolle der intraoperativen Cholangiografie bei der laparoskopischen Cholezystektomie

Nach Einführung der laparoskopischen Cholezystektomie ist die Rolle der früher bei der offenen Cholezystektomie praktisch obligaten intraoperativen Cholangiografie stark relativiert worden. In verschiedenen Untersuchungen und Umfragen wird deutlich, dass die Anwendung der routinemäßigen intraoperativen Cholangiografie heute bei weit unter 10 % der laparoskopischen Cholezystektomien liegt. Im Zeitraum von 1991 - 2000 wurden insgesamt 1.573 Patienten aufgrund einer symptomatischen Cholezystolithiasis laparoskopisch operiert, 1.311 davon mit IOC und 262 ohne IOC (überwiegend unmittelbar nach Einführung der laparoskopischen Operationsmethode.) Die Erhebung der Daten erfolgte retrospektiv. Auf Grund der Auswertung der vorliegenden Daten und der Auswertung der Literatur sollte die IOC nicht nur selektiv, sondern routinemäßig bei jeder laparoskopischen Cholezystektomie durchgeführt werden. Nur in drei Fällen (von 1.573 erfolgten Cholezystektomien) erfolgte eine irrtümliche Durchtrennung des Ductus choledochus. Diese konnte durch die IOC unmittelbar diagnostiziert und entsprechend durch Reanastomosierung behandelt werden. Der oft komplizierte Verlauf nach verschleppter Diagnostik führt häufig zu entzündlichen Komplikationen bis hin zu einzelnen Todesfällen und macht meist die Anlage einer biliodigestiven Anastomose notwendig. Eine erhöhte Operationsmorbidität und eingeschränkt gute Langzeitergebnisse begründen die große Bedeutung der rechtzeitigen Diagnostik und konsequenten Therapie einer Choledochusläsion. Auch bei der akuten Cholezystitis mit oft schwer entzündlich veränderten anatomischen Strukturen, welche die intraoperative Darstellung der Anatomie erheblich erschwert, führt eine routinemäßige IOC ebenfalls zur Reduktion von iatrogenen Verletzungen

LASER treatment in gynaecology -A randomized controlled trial in women with symptomatic lichen sclerosus.

OBJECTIVE Aim of our study was to evaluate the therapeutic effect of laser treatment in vulvar lichen sclerosus, mainly the reduction of existing symptoms as itching, burning and pain. We asked about the different outcome by using different application doses. STUDY DESIGN We conducted a prospective randomized double-blind dose-controlled trial in our dysplasia unit specializing vulvar disorders. 67patients with active LS were included. LS was confirmed by biopsy or by the validated CSS (clinical scoring system of vulvar LS). Computer generated randomization resulted in two groups, each group received a different application dose.(LDG- low dose group, NDG- normal dose group) During the study period of 18 weeks all participants received three laser applications in three subsequent sessions of three weeks. Two follow-ups six and twelve weeks after the first application was performed. At every visit, the participants filled in the VAS (visual analogue scale) for recording the actual vulvar symptoms as itching burning or pain on a range from 0 to 10. RESULTS Before treatment the mean VAS-Score was 4.3 (STD ± 2.4) in the NDG and 5.1(±2.6) in the LDG. After 18 weeks, the mean reduction was -2.4 (±2.3) for NDG and -2.7 (±2.8) for LDG. Four patients (two of each group) reported more pain after than before treatment. Both groups show significant lower VAS-Scores 18 weeks after the treatment than before therapy (p < 0.0001). The reduction of symptoms after 18 weeks between NDG and LDG was not significant (p = 0.6244). CONCLUSION Laser treatment with the microablative CO2 laser leads to a significant improvement for symptoms of LS. A higher dosage of laser radiation shows no benefit concerning the symptoms. We have not observed any serious adverse events during this study

Efficacy of topical progesterone versus topical clobetasol propionate in patients with vulvar Lichen sclerosus - A double-blind randomized phase II pilot study.

BACKGROUND Lichen sclerosus (LS) is a chronic inflammatory skin disease that mostly affects the anogenital region of women and lowers patients' quality of life. Current standard treatment of LS is topical steroids. OBJECTIVE To evaluate the efficacy of topical progesterone 8% ointment and compare to standard therapy with topical clobetasol propionate 0.05% in premenopausal women presenting with previously untreated early onset LS. STUDY DESIGN Randomized, double-blind, 2-arm, single center superiority trial in premenopausal women with histologically confirmed vulvar LS who were randomized in a 1:1 ratio to receive clobetasol propionate 0.05% ointment or progesterone 8% ointment. The primary outcome was the clinical severity LS score after 12 weeks, which consists of six clinical features assessed by the physician. Secondary outcomes were the symptom severity LS score, which consists of three symptoms rated by the patient, the Short Form SF-12 physical and mental health scores, and adverse events. Response to medication was assessed by biopsy at the end of the treatment to evaluate inflammatory parameters. RESULTS Overall, 105 women were screened, 102 underwent vulvar biopsy and 37 received a histologically confirmed diagnosis of LS and were randomized: 17 to progesterone and 20 to clobetasol propionate. At 12 weeks, the mean clinical LS scores improved from 4.6 (SD 2.0) to 4.5 (SD 1.7) in the progesterone arm, and from 4.6 (SD 2.8) to 2.9 (SD 2.2) in the clobetasol propionate arm (difference in favor of clobetasol 1.61; 95% CI 0.44 to 2.77, p = 0.009), and the mean symptom severity LS scores improved from 4.5 (SD 3.8) to 3.1 (SD 3.0) in the progesterone arm, and from 4.7 (SD 2.8) to 1.9 (SD 1.8) in the clobetasol propionate arm (difference in favor of clobetasol 1.32; 95% CI -0.25 to 2.89, p = 0.095). LS was in complete remission in 6 out of 10 patients (60%) with available biopsy in the progesterone arm, and in 13 out of 16 patients (81.3%) in the clobetasol propionate arm (odds ratio in favor of clobetasol 0.35; 95% CI 0.06 to 2.06, p = 0.234). No drug-related serious adverse event occurred during the trial. CONCLUSIONS Topical progesterone 8% ointment is inferior to standard therapy with topical clobetasol propionate 0.05% in previously untreated premenopausal women with vulvar LS after 12 weeks treatment

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

<p>Abstract</p> <p>Background</p> <p>The parvulin-type peptidyl prolyl <it>cis/trans </it>isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study.</p> <p>Results</p> <p>Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions.</p> <p>Conclusion</p> <p>Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae.</p

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR

Aim: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). Materials & methods: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. Results: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. Conclusion: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR. © 2018 Daniel Weiss

Indications and practical approach to non-invasive ventilation in acute heart failure

In acute heart failure (AHF) syndromes significant respiratory failure (RF) is essentially seen in patients with acute cardiogenic pulmonary oedema (ACPE) or cardiogenic shock (CS). Non-invasive ventilation (NIV), the application of positive intrathoracic pressure through an interface, has shown to be useful in the treatment of moderate to severe RF in several scenarios. There are two main modalities of NIV: continuous positive airway pressure (CPAP) and pressure support ventilation (NIPSV) with positive end expiratory pressure. Appropriate equipment and experience is needed for NIPSV, whereas CPAP may be administered without a ventilator, not requiring special training. Both modalities have shown to be effective in ACPE, by a reduction of respiratory distress and the endotracheal intubation rate compared to conventional oxygen therapy, but the impact on mortality is less conclusive. Non-invasive ventilation is also indicated in patients with AHF associated to pulmonary disease and may be considered, after haemodynamic stabilization, in some patients with CS. There are no differences in the outcomes in the studies comparing both techniques, but CPAP is a simpler technique that may be preferred in low-equipped areas like the pre-hospital setting, while NIPSV may be preferable in patients with significant hypercapnia. The new modality 'high-flow nasal cannula' seems promising in cases of AHF with less severe RF. The correct selection of patients and interfaces, early application of the technique, the achievement of a good synchrony between patients and the ventilator avoiding excessive leakage, close monitoring, proactive management, and in some cases mild sedation, may warrant the success of the technique

Evaluation of Clinical Risk Factors to Predict High On-Treatment Platelet Reactivity and Outcome in Patients with Stable Coronary Artery Disease (PREDICT-STABLE)

Objectives This study was designed to identify the multivariate effect of clinical risk factors on high on-treatment platelet reactivity (HPR) and 12 months major adverse events (MACE) under treatment with aspirin and clopidogrel in patients undergoing non-urgent percutaneous coronary intervention (PCI). Methods 739 consecutive patients with stable coronary artery disease (CAD) undergoing PCI were recruited. On-treatment platelet aggregation was tested by light transmittance aggregometry. Clinical risk factors and MACE during one-year follow-up were recorded. An independent population of 591 patients served as validation cohort. Results Degree of on-treatment platelet aggregation was influenced by different clinical risk factors. In multivariate regression analysis older age, diabetes mellitus, elevated BMI, renal function and left ventricular ejection fraction were independent predictors of HPR. After weighing these variables according to their estimates in multivariate regression model, we developed a score to predict HPR in stable CAD patients undergoing elective PCI (PREDICT-STABLE Score, ranging 0-9). Patients with a high score were significantly more likely to develop MACE within one year of follow-up, 3.4% (score 0-3), 6.3% (score 4-6) and 10.3% (score 7-9); odds ratio 3.23, P=0.02 for score 7-9 vs. 0-3. This association was confirmed in the validation cohort. Conclusions Variability of on-treatment platelet function and associated outcome is mainly influenced by clinical risk variables. Identification of high risk patients (e.g. with high PREDICT-STABLE score) might help to identify risk groups that benefit from more intensified antiplatelet regimen. Additional clinical risk factor assessment rather than isolated platelet function-guided approaches should be investigated in future to evaluate personalized antiplatelet therapy in stable CAD-patients

Acute Heart Failure in the 2021 ESC Heart Failure Guidelines: a scientific statement from the Association for Acute CardioVascular Care (ACVC) of the European Society of Cardiology.

The current European Society of Cardiology (ESC) Heart Failure Guidelines are the most comprehensive ESC document covering heart failure to date; however, the section focused on acute heart failure remains relatively too concise. Although several topics are more extensively covered than in previous versions, including some specific therapies, monitoring and disposition in the hospital, and the management of cardiogenic shock, the lack of high-quality evidence in acute, emergency, and critical care scenarios, poses a challenge for providing evidence-based recommendations, in particular when by comparison the data for chronic heart failure is so extensive. The paucity of evidence and specific recommendations for the general approach and management of acute heart failure in the emergency department is particularly relevant, because this is the setting where most acute heart failure patients are initially diagnosed and stabilized. The clinical phenotypes proposed are comprehensive, clinically relevant and with minimal overlap, whilst providing additional opportunity for discussion around respiratory failure and hypoperfusion.F.P. has received research grants from Abbott, Becton Dickenson, Brainbox, Calcimedica, CSL Behring, Cue, Ortho Clinical Diagnostics, Relypsa, Roche, Salix, Siemens. Consultant: Abbott, Astra-Zeneca, Beckman, Bosch, Fast Biomedical, Forrest Devices, Ischemia Care, Dx, Instrument Labs, Janssen, Nabriva, Ortho Clinical Diagnostics, Osler, Relypsa, Roche, Quidel, Salix, Siemens, Upstream and has stock/ownership interests in AseptiScope Inc, Brainbox Inc, Braincheck Inc, Coagulo Inc, Comprehensive Research Associates LLC, Comprehensive Research Management Inc, Emergencies in Medicine LLC, Fast Inc, Forrest Devices, Ischemia DX LLC, Lucia Inc, Prevencio Inc, ScPharma, Trivirum Inc, Upstream Inc. E.P.’ employer has received support from Novartis for consulting work and she has consulted for scPharmaceuticals outside of the submitted work. She has received research support from the NIH (R01HL148439).S