Efficacy of sulbactam in an in vitro model of mixed aerobic/anaerobic infections

Summary Bacterial interactions in mixed infections may interfere with antimicrobial therapy. Thein-vitro efficacy of ampicillin alone, combination sulbactam/ampicillin, and metronidazole was studied. Strains ofBacteroides fragilis, Escherichia coli, andEnterococcus faecalis, alone and in association, were tested by means of a broth dilution method. Minimal bactericidal concentrations (MBC) of ampicillin forB. fragilis 74 in association withE. coli 68 were up to 16-fold higher than forB. fragilis 74 alone (256 compared to 16 mg/l), but only 2-fold higher for sulbacta(5 mg/l)/ampicillin (0.5 and 0.25 g/l). Association ofB. fragilis 45 andE. faecalis 186 increased ampicillin MBC ofE. faecalis 186 from 2 to 16 mg/l, but the combination sulbactam/ampicillin restored activity of ampicillin. In association withE. faecalis, metronidazole MBCs ofB. fragilis increased up to 64-fold. Strains ofE. faecalis andE. coli were able to destroy 10 mg/l metronidazole within 8 to 20 h. The present experiments demonstrated effectiveness of sulbactam/ampicillin to inhibit -lactamases of associated pathogens. Destruction of metronidazole byE. faecalis lends additional support to the use of the combination in aerobic/anaerobic infections includingE. faecalis.

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In vitro-Wirksamkeit von Sulbactam in einem Modell für aerob/anaerobe Mischinfektionen

Zusammenfassung Bakterielle Interaktionen in Mischinfektionen können die Wirksamkeit einer antibiotischen Therapie beeinflussen. DieIn-vitro-Wirksamkeit von Ampicillin, Sulbactam/Ampicillin sowie von Metronidazol wurde mit Stämmen vonBacteroides fragilis, Escherichia coli undEnterococcus faecalis einzeln und in Association mittels einer Bouillonverdünnungsmethode getestet. Die minimale bakterizide Konzentration (MBK) von Ampicillin fürB. fragilis 74 in Association mitE. coli 68 war bis zu 16 mal höher als bei der Testung vonB. fragilis 74 alleine (256 mg/l bzw. 16 mg/l), zweimal höher mit Sulbactam (5 mg/l)/Ampicillin (0,5 und 25 mg/l). In Assoziation mitB. fragilis 45 und erhöhte sich die Ampicillin-MBK fürE. faecalis 186 von 2 auf 16 mg/l, die Kombination mit Sulbactam stellte die Wirksamkeit von Ampicillin wieder her. Die Metronidazol-MBK-Werte fürB. fragilis stiegen in Assoziation mitE. faecalis um das bis zu 64fache an.E. faecalis- undE. coli-Stämme waren in der Lage, 10 mg/l Metronidazol innerhalb von 8 bis 20 h abzubauen. Die vorliegenden Experimente demonstrierten die Wirksamkeit von Sulbactam/Ampicillin bei der Hemmung von -Laktamasen assoziierter Erreger. Auch bei aerob/anaeroben Infektionen mit Beteiligung vonE. faecalis kann diese Kombination vorteilhaft sein.

     

The PCBP1 gene encoding poly(rc) binding protein i is recurrently mutated in Burkitt lymphoma

The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG‐MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole‐genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron‐less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K‐Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre‐mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20–40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis. © 2015 Wiley Periodicals, Inc.     

Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.Worldwide, more than 5 million babies (Ferraretti et al. 2013) have been born through in vitro fertilization (IVF) since the birth of the first in 1978 (Steptoe and Edwards 1978). Exact numbers are difficult to determine, but it has been estimated that currently 350,000 babies are born yearly through IVF (de Mouzon et al. 2009, 2012; Centers for Disease Control and Prevention 2011; Ferraretti et al. 2013). That number is expected to rise, as advanced maternal age is associated with decreased fertility rates and women in developed countries continue to delay childbirth to later ages. In 95% of IVF procedures, no diagnostic testing of the embryos is performed (https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx?ClinicPKID=0). Couples with prior difficulties conceiving or those wishing to avoid the transmission of highly penetrant heritable diseases often choose to perform preimplantation genetic diagnosis (PGD). PGD involves the biopsy of one cell from a 3-d embryo or the recently more preferred method, due to improved implantation success rates (Scott et al. 2013b), of up to 10 cells from a 5- to 6-d blastocyst-stage embryo. Following biopsy, genetic analysis is performed on the isolated cell(s). Currently this is an assay for translocations and the correct chromosome copy number (Hodes-Wertz et al. 2012; Munne 2012; Yang et al. 2012; Scott et al. 2013a; Yin et al. 2013), a unique test designed and validated for each specific heritable disease (Gutierrez-Mateo et al. 2009), or a combination of both (Treff et al. 2013). Importantly, none of these approaches can detect de novo mutations.Advanced maternal age has long been associated with an increased risk of producing aneuploid embryos (Munne et al. 1995; Crow 2000; Hassold and Hunt 2009) and giving birth to a child afflicted with Down syndrome or other diseases resulting from chromosomal copy number alterations. Conversely, children of older fathers have been shown to have an increase in single base and short multibase insertion/deletion (indels) de novo mutations (Kong et al. 2012). Many recent large-scale sequencing studies have found that de novo variations spread across many different genes are likely to be the cause of a large fraction of autism cases (Michaelson et al. 2012; O’Roak et al. 2012; Sanders et al. 2012; De Rubeis et al. 2014; Iossifov et al. 2014), severe intellectual disability (Gilissen et al. 2014), epileptic encephalopathies (Epi4K Consortium and Epilepsy Phenome/Genome Project 2013), and many other congenital disorders (de Ligt et al. 2012; Veltman and Brunner 2012; Yang et al. 2013; Al Turki et al. 2014). Additionally rare and de novo variations have been suggested to be prevalent in patients with schizophrenia (Fromer et al. 2014; Purcell et al. 2014), and Michaelson et al. (2012) found that single base de novo mutations affect conserved regions of the genome and essential genes more often than regions of unknown function. Current targeted approaches to PGD would miss many of these important functional changes within the embryonic DNA sequence, and even a whole-genome sequencing (WGS)–based carrier screen of both parents would not enable comprehensive preimplantation or prenatal diagnoses due to de novo mutations. As more parents delay childbirth into their mid-30s and later, these studies suggest we should try to provide better diagnostic tests for improving the health of newborns. In this study, we demonstrate the use of an advanced WGS process that provides an accurate and phased genome sequence from about 10 cells, allowing highly sensitive and specific detection of single base de novo mutations from IVF blastocyst biopsies.     

Bestrophin 1 – Phenotypes and Functional Aspects in Bestrophinopathies

This is to review the current state of knowledge on the functional and clinical aspects of bestrophin 1, a prominent member of a family of proteins involved in the control and properties of the light peak of the EOG. Initially human bestrophin 1 gene (BEST1) mutations were identified to underlie Best vitelliform macular dystrophy (VMD), a dominantly inherited, juvenile-onset form of macular degeneration. In the recent past the phenotypical spectrum of retinal disorders associated with BEST1 mutations has been extended and the term bestrophinopathies was coined. The physiological role of bestrophin 1 is still not completely understood but has been linked to the generation of a transepithelial chloride current by controlling voltage-dependent calcium channels (VDCC). Dysfunction of bestrophin 1 may result in abnormal ion and fluid transport by the retinal pigment epithelium (RPE) disturbing and even disrupting direct interactions between the RPE and the photoreceptors.     

Separating stimulus‐driven and response‐related LRP components with Residue Iteration Decomposition (RIDE)

When the lateralized readiness potential (LRP) is recorded in stimulus–response compatibility (SRC) tasks, two processes may overlap in the LRP, stimulus‐driven response priming and activation based on response selection rules. These overlapping processes are hard to disentangle with standard analytical tools. Here, we show that Residue Iteration Decomposition (RIDE), based on latency variability, separates the overlapping LRP components from a Simon task into stimulus‐driven and response‐related components. SRC affected LRP amplitudes only in the stimulus‐driven component, whereas LRP onsets were affected only in the response‐locked component. Importantly, the compatibility effect in reaction times was more similar to the effect in the onsets of the RIDE‐derived response‐locked LRP component than in the unseparated LRP. Thus, RIDE‐separated LRP components are devoid of distortions inherent to standard LRPs.