Background: Quorum sensing is the mechanism by which bacteria control gene expression in response to cell density. Two major quorum-sensing systems have been identified, system 1 and system 2, each ...with a characteristic signaling molecule (autoinducer-1, or AI-1, in the case of system 1, and AI-2 in system 2). The
luxS gene is required for the AI-2 system of quorum sensing. LuxS and AI-2 have been described in both Gram-negative and Gram-positive bacterial species and have been shown to be involved in the expression of virulence genes in several pathogens.
Results: The structure of the LuxS protein from three different bacterial species with resolutions ranging from 1.8 Å to 2.4 Å has been solved using an X-ray crystallographic structural genomics approach. The structure of LuxS reported here is seen to have a new alpha-beta fold. In all structures, an equivalent homodimer is observed. A metal ion identified as zinc was seen bound to a Cys-His-His triad. Methionine was found bound to the protein near the metal and at the dimer interface.
Conclusions: These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.
Recent technological advances have greatly increased both the speed and throughput of genome and transcriptome sequencing. The pace of sequencing has further increased with target-enrichment and ...library barcoding techniques that allow multiplexing of sequencing runs. However, current manual methods for creating libraries do not scale well, limiting the practical investigation of large numbers of samples. To ease the library-creation bottleneck, we describe here a set of protocols, robotics scripts, bulk reagents and instrumentation developed to automate the production of up to 96 DNA fragment sequencing libraries at once. Current protocols for creating sequencing libraries are lengthy, laborious, and not amenable to automation. We describe here new magnetic bead based methods that produce libraries with yield, purity, and size-selection comparable or superior to current column and gel-based protocols. Using these bead-based methods we developed a unique combination of optimized adaptor concentrations and clean-up techniques which increase the yield of libraries from small amounts of DNA by several fold. We also describe robotic scripts for producing 1–96 libraries simultaneously on two commonly used robotic platforms. These protocols accept 10–8000ng of sheared DNA, calculate and dilute barcode adaptors for each library as necessary, automate all intermediate processing steps, and deliver purified libraries ready for amplification off-instrument. An additional script for post-PCR purification of the libraries is also provided. We further describe the Library Builder™ System, comprising a bench top device and kits of plastic tips, tubes and sealed cartridges prefilled with reagents necessary for producing 1 to 13 DNA fragment libraries either with or without size selection. Our analysis of sequencing data shows that libraries produced by all of the above methods are free of excess adaptors which interfere with quantitation, are unbiased, of high complexity, and free from cross-contamination. The protocols herein are provided to the community for use or customization.
Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral ...producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.
BACKGROUND:The occurrence of anastomotic leakage is still a life-threatening complication for patients after colorectal surgery. In literature not only an impact on the short-term outcome but also on ...long-term survival and local recurrence of colorectal cancer patients is discussed.
OBJECTIVE:This study aimed to investigate the impact of anastomotic leakage on long-term survival and local recurrence.
DESIGN:A total of 1122 patients with resections for colorectal cancer were analyzed. In 94 patients (8.4%) there was clinical proof of anastomotic leakage. A reference group was defined as the 1028 patients without anastomotic leakage using 1:1 propensity score-matching according to the following criteriaage, sex, International Union Against Cancer stage, Karnofsky index, tumor site, and grading, as well as adjuvant chemotherapy. A calculation of overall survival, disease-free survival, and local recurrence rate was performed for both groups.
SETTINGS:The study was conducted using a retrospective matched-pairs analysis, based on a prospectively maintained institutional colorectal cancer database.
PATIENTS:Ninety-four patients with anastomotic leakage and 94 matched control subjects from a total of 1122 patients with resections for colorectal cancer were studied.
MAIN OUTCOME MEASURES:Overall survival, disease-free survival, and local recurrence rate for patients with colorectal cancer with and without anastomotic leakage were measured.
RESULTS:The propensity score matching successfully created 2 groups with no significant differences in the matching criteria. Survival analysis disclosed no significant differences between the groups in terms of overall survival, disease-free survival, and local recurrence rate. Univariate analysis identified age, Karnofsky index, International Union Against Cancer stage, and lymph node metastasis as significant prognostic factors. Multivariable analysis of these variates revealed age and positive lymph nodes as independent predictors of overall survival and disease-free survival.
LIMITATIONS:The study was limited by nature of being a retrospective analysis and monocentric study.
CONCLUSIONS:This matched-pairs analysis, comparing patients with colorectal cancer with and without anastomotic leakage, revealed no significant differences in overall survival, disease-free survival, and local recurrence rate. Contrary results in the literature might be caused by nonbalanced settings in nonmatched collectives. See Video Abstract at http://links.lww.com/DCR/A811.
The inflammatory and immune systems are altered in type 2 diabetes. Here, the aim was to profile the immune and inflammatory response in subjects with prediabetes and diabetes in a large ...population-representative sample.
In total, 15,010 individuals were analyzed from the population-based Gutenberg Health Study. Glucose status was classified according to HbA1c concentration and history of diagnosis. All samples were analyzed for white blood cells (WBCs), granulocytes, lymphocytes, monocytes, platelets, C-reactive protein (CRP), albumin, fibrinogen, and hematocrit. Interleukin-18 (IL-18), IL-1 receptor antagonist (IL-1RA), and neopterin concentrations were determined in a subcohort.
In total, 7,584 men and 7,426 women were analyzed (range 35-74 years), with 1,425 and 1,299 having prediabetes and diabetes, respectively. Biomarkers showed varying dynamics from normoglycemic via subjects with prediabetes to subjects with diabetes: 1) gradual increase (WBCs, granulocytes, monocytes, IL-1RA, IL-18, and fibrinogen), 2) increase with subclinical disease only (lymphocytes and CRP), 3) increase from prediabetes to diabetes only (neopterin), and 4) no variation with glucose status (hematocrit). The strongest relative differences were found for CRP, IL-1RA, and fibrinogen concentrations. Several inflammatory and immune markers were associated with the glucose status independent from cardiovascular risk factors and comorbidities, varied with disease severity and the presence of disease-specific complications in the diabetes subcohort.
The inflammatory and immune biomarker profile varies with the development and progression of type 2 diabetes. Markers of inflammation and immunity enable differentiation between the early preclinical and clinical phases of the disease, disease complications, and progression.
To describe the distribution of intraocular pressure (IOP) and its association with ocular features and cardiovascular risk factors in an adult European cohort.
Population-based, cross-sectional ...study.
This analysis was based on a Gutenberg Health Study (GHS) cohort that included 4335 eligible enrollees from among 5000 subjects who participated in the survey from 2007 through 2008. The age range was 35 to 74 years at enrollment.
Participants underwent a standardized protocol with a comprehensive questionnaire; ophthalmic examination including slit-lamp biomicroscopy, noncontact tonometry, fundus photography, central corneal thickness measurement, and visual field testing; and a thorough general examination focused on cardiovascular parameters, psychological evaluation, and laboratory tests, including genetic analysis.
Mean and reference interval of IOP stratified by age, gender, and eye.
Mean ± standard deviation (SD) IOP was 14.0 ± 2.6 mmHg in both eyes, 13.9 ± 2.7 mmHg in right eyes, and 14.0 ± 2.7 mmHg in left eyes. Mean ± SD IOP in men (n = 2216) and in women (n = 2119) was 14.1 ± 2.7 mmHg and 13.9 ± 2.5 mmHg with an intersex difference (P = 0.009). Positive univariate associations with higher IOP were detected for brown iris color, central corneal thickness, hypertension, diabetes, smoking, obesity, dyslipidemia, body mass index, weight, hip size (women only), waist circumference, and waist-to-hip ratio. Multivariate testing revealed male gender, central corneal thickness, brown iris color, hypertension, smoking, and waist-to-hip ratio to be correlated with higher IOP. In women, age correlated negatively with IOP in the multivariate analysis.
Intraocular pressure distribution in this cohort yielded a lower mean IOP than in similar white study populations. Increasing age in women correlated with lower IOP. Association analyses with several systemic characteristics revealed that cardiovascular risk factors correlated with higher IOP.
The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Hintergrund:
Die Lernkurve der minimal-invasiven Chirurgie insbesondere in der Thoraxchirurgie ist durch hohe Anforderungen charakterisiert. Standardisierte Trainingscurricula, v.a. zum Erlernen der ...Fertigkeiten außerhalb des Operationssaals, existieren weitgehend bisher nicht.
Material und Methoden:
Das Gesamtkonzept der Lübecker Toolbox umfasst eine Trainingsbox, sechs Übungsmodule, Zielvorgaben für die Übungen, Empfehlungen für einen effektiven Trainingsablauf und didaktisches Videomaterial. Die Standardisierung der Zielvorgaben erfolgte durch die Erfassung der Leistung von 15 erfahrenen laparoskopisch tätigen Chirurgen. Die Mittelwerte der Bestzeiten wurden als Benchmark für eine prospektive Studie mit 30 MIC-unerfahrenen Probanden festgelegt. Vor und nach Abschluss des Trainingscurriculums absolvierten alle Probanden eine Cholezystektomie am Organpaket, die jeweils videodokumentiert und verblindet durch zwei Chirurgen mittels des GOALS-Scores bewertet wurde.
Ergebnisse:
Alle Probanden schlossen die Studie ab. Die Mittelwerte der Bestzeiten durch die Erfahrenen betrugen 72 ± 8sec, 49 ± 9 s, 66 ± 10sec, 89 ± 28sec und 138 ± 44sec. Die Anzahl an Wiederholungen zum Erreichen der Zielvorgabe für die 6 Übungen lag im Median bei 42 (7 – 80), 26 (9 – 55), 32 (14 – 77), 44 (15 – 59), 19 (6 – 68) und 26 (15 – 60). Bei einer Übungsdauer von 30 min/Einheit und 5 bzw. 7 Übungseinheiten/Woche, sind im Median 37 (23 – 49) Übungseinheiten bzw. 5 – 7 Wochen zur Komplettierung des Curriculums erforderlich. Der GOALS-Score besserte sich signifikant nach Abschluss des Trainingscurriculums.
Schlussfolgerung:
Mit der „Lübecker Toolbox“ wurde ein standardisiertes Trainingscurriculum für die MIC konzipiert. Die erlernten Fertigkeiten gehören zur minimal-invasiven Basisausbildung aller mit dieser Methode befassten chirurgischen Fachgebiete. Eine standardisierte Einführung in der minimal-invasiven Chirurgie kann die Ausbildung in den Fächern Viszeralchirurgie, Thoraxchirurgie und Gynäkologie verbessern und beschleunigen.
Hintergrund:
Seit langem wird angenommen, dass Aneuploidie einen unabhängigen Prognoseparameter für das kolorektale Karzinom (KRK) darstellt. Aufgrund der Heterogenität der zahlreichen Studien wird ...die Bestimmung des Ploidiestatus derzeit für die Routinediagnostik nicht empfohlen. In Hinblick auf die Frage, ob in vielen Studien ein Bias aufgrund stadienspeziefischer Differenzen im Auftreten der Aneuploidiebesteht, führten wir eine Metaanalyse und einen systematischen Review durch.
Methodik:
Ein systematischer Internet-basierter Suchprozess ergab 1.935 in Englisch und zwischen 1990 und 2011 publizierte Studien. Der definierte Endpunkt unserer Metaanalyse war ein Anstieg der Raten an Aneuploidie zwischen frühen (Dukes A+B/UICC I+II, n = 3.632 Proben) und fortgeschrittenen Stadien des KRK (Dukes C+D/UICC III+IV, n = 3.440 Proben.
Ergebnisse:
Von den 1.935 initial identifizierten Studien wurden 17 Studien zur Bildzytometrie (n = 2.130 Patienten) und 20 Studien zur Durchflusszytometrie (n = 7.023 Patienten) im Detail untersucht. Die Metaanalyse ergab, dass fortgeschrittene KRK signifikant häufiger Aneuploidie aufwiesen als die frühen KRK (n = 7.072 Patienten, odds ratio 1,51; 95%-CI 1,37 – 1,67; p = 0,0007). Unabhängig vom Tumorstadium betrug die Rate der Aneuploidie zwischen 39% und 81% (Median 58%) und insgesamt beschrieben 21 Studien (54,1%) eine prognsotische Signifikanz für die Aneuploidie bezüglich Gesamt-, tumorspeziefischem oder rezidivfreiem Überleben.
Schlussfolgerungen:
Die Zunahme der Rate an Aneuploidie in fortgeschrittenen Stadien des KRK deutet auf eine Zunahme der genomischen Instabilität im Rahmen der Tumorprogression hin. Während ein Großteil der Studien eine prognostische Relevanz der Aneuploidie beschrieb, konnten nur wenige Studien die Aneuploidie auch als stadienspeziefischen Marker identifizieren. Die Daten unserer Analyse betonen die Notwendigkeit, multizentrische Studien mit standardisierten Techniken und Definitionen zur Ploidiebestimmung durchzuführen, um die prognostische Relevanz der Aneuploidie auch bezüglich individueller Tumorstadien zu klären.
PDF
