A global view on carbapenem-resistant Acinetobacter baumannii.

To give an update on the molecular epidemiology and global distribution of carbapenemase encoding genes, we subjected 313 carbapenem-resistant Acinetobacter baumannii isolated from 114 study centers in 47 countries in five world regions, Africa, Asia, Europe, Latin America, and North America, to whole genome sequencing. Numbers of isolates investigated were proportional to the population size of the contributing countries. Molecular epidemiology was investigated using seven-loci and core genome multilocus sequence typing, whole-genome single nucleotide polymorphism phylogenies, and the intrinsic blaOXA-51-like variant. Carbapenemase encoding genes were identified by multiplex PCR and ResFinder. Among the total of 313 isolates, 289 (92.3%) were assigned to A. baumannii international clones (IC) IC1-IC8. IC2 predominated with 196 isolates (62.6%) and was spread worldwide, followed by IC5 with 44 isolates (14.1%) mainly confined to Latin America. Six isolates (1.9%) originating from Belgium, Egypt, Italy, and Pakistan represent the novel IC9. Acquired OXA-type carbapenemase genes were found in 300 (96%) isolates with blaOXA-23-like and blaOXA-40-like predominating, which constitutes a significant increase compared to our findings from 2010. Metallo-beta-lactamases were rare with seven isolates (2.2%). The distribution of ICs and carbapenemase determinants can vary widely among different geographical regions. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii are of increasing public health importance, as they are resistant to last-line antibiotics. International clones with well-characterized resistance genes dominate globally; however, locally, other lineages with different properties may be of importance to consider. This study investigated isolates from a broad geographic origin from 114 hospitals in 47 countries and from five world regions ensuring the greatest possible diversity in an organism known for its propensity for clonal epidemic spread and reflecting the current global epidemiology of carbapenem-resistant A. baumannii. In Latin America, a lineage different from other geographic regions circulates, with a different resistance gene profile. This knowledge is important to adjust local infection prevention measures. In a global world with migration and increasing use of antimicrobials, multidrug-resistant bacteria will continue to adapt and challenge our healthcare systems worldwide.

In Vitro Activity of Cefiderocol against a Global Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates.

Background: Cefiderocol is a novel siderophore cephalosporin with potent activity against multi-drug-resistant Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii (CRAB). Methods: The susceptibility of 313 non-duplicate CRAB isolates with defined carbapenem resistance mechanisms from a global collection to cefiderocol, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, ciprofloxacin, colistin, imipenem/relebactam, meropenem, meropenem/vaborbactam, minocycline, and piperacillin/tazobactam was determined using the broth microdilution method. Isolates were obtained from various body sites from patients in 47 countries in five world regions between 2012 and 2016. The identification of carbapenem resistance mechanisms and assignment to A. baumannii international clonal lineages were based on whole genome sequencing. Results: Cefiderocol showed greater activity than comparator antimicrobials of the ß-lactam class, including novel ß-lactams combined with ß-lactamase inhibitors, ciprofloxacin, and minocycline. Cefiderocol MIC50 and MIC90 values were 0.5 mg/L and 4 mg/L, respectively, while colistin had comparable activity with a higher MIC50 at 1 mg/L and a lower MIC90 value of 2 mg/L. Many isolates with elevated cefiderocol MICs ≥ 4 mg/L represented A. baumannii international clone (IC) 1 and harbored a metallo-ß-lactamase. Conclusions: While cefiderocol is a useful addition to the limited armamentarium of drugs targeting this problematic pathogen, a considerable part of CRAB isolates had elevated MIC values in a range of 4 -> 32 mg/L, including all isolates with a metallo-ß-lactamase.

The Unified Redox Scale for All Solvents: Consistency and Gibbs Transfer Energies of Electrolytes from their Constituent Single Ions.

We have devised the unified redox scale Eabs H2O , which is valid for all solvents. The necessary single ion Gibbs transfer energy between two different solvents, which only can be determined with extra-thermodynamic assumptions so far, must clearly satisfy two essential conditions: First, the sum of the independent cation and anion values must give the Gibbs transfer energy of the salt they form. The latter is an observable and measurable without extra-thermodynamic assumptions. Second, the values must be consistent for different solvent combinations. With this work, potentiometric measurements on silver ions and on chloride ions show that both conditions are fulfilled using a salt bridge filled with the ionic liquid [N2225 ][NTf2 ]: if compared to the values resulting from known pKL values, the silver and chloride single ion magnitudes combine within a uncertainty of 1.5 kJ mol-1 to the directly measurable transfer magnitudes of the salt AgCl from water to the solvents acetonitrile, propylene carbonate, dimethylformamide, ethanol, and methanol. The resulting values are used to further develop the consistent unified redox potential scale Eabs H2O that now allows to assess and compare redox potentials in and over six different solvents. We elaborate on its implications.

Molecular diagnostics enables detection of actionable targets: the Pediatric Targeted Therapy 2.0 registry.

BACKGROUND: Precision oncology requires diagnostic accuracy and robust detection of actionable alterations. The Pediatric Targeted Therapy (PTT) 2.0 program aims at improving diagnostic accuracy by addition of molecular analyses to the existing histological diagnosis and detection of actionable alterations for relapsed paediatric oncology patients, in cases with limited availability of tumour material. METHODS: Paediatric patients diagnosed with relapse or progression of a central nervous system tumour (n = 178), a sarcoma (n = 41) or another solid tumour (n = 44) were included. DNA methylation array, targeted gene panel sequencing on tumour and blood (130 genes), RNA sequencing in selected cases and a pathway-specific immunohistochemistry (IHC) panel were performed using limited formalin-fixed paraffin embedded tissue from any disease episode available. The clinical impact of reported findings was assessed by a serial questionnaire-based follow-up. RESULTS: Integrated molecular diagnostics resulted in refined or changed diagnosis in 117/263 (44%) tumours. Actionable targets were detected in 155/263 (59%) cases. Constitutional DNA variants with clinical relevance were identified in 16/240 (7%) of patients, half of which were previously unknown. Clinical follow-up showed that 26/263 (10%) of patients received mechanism-of-action based treatment matched to the molecular findings. CONCLUSION: Next-generation diagnostics adds robust and relevant information on diagnosis, actionable alterations and cancer predisposition syndromes even when tissue from the current disease episode is limited.

The sweet tooth of infancy: Is sweetness exposure related to sweetness liking in infants up to 12 months of age?

Infants become increasingly exposed to sweet-tasting foods in their first year of life. However, it is still unclear whether repeated exposure to sweet taste is linked to infants' sweetness liking during this period. Making use of data from the OPALINE cohort, this study aimed to examine the link between sweetness exposure and sweetness liking during two important periods in early infant feeding: at the start of complementary feeding (3-6 months) and the transition to the family table (10-12 months). Infants' sweetness exposure was assessed using 7-d food records which were completed by mothers every month (n 312), reporting daily consumption rates of formula/breast milk or complementary food and the type of formula milk and/or complementary foods for each feeding occasion. Infants' sweetness liking was studied in the laboratory at 3, 6 and 12 months of age by assessing their response to a lactose-water solution and the amount drunk of this solution compared with plain water. Linear regressions and structural equation model assessed associations between exposure to and liking for sweetness at 6 and 12 months. Neither at 6 (n 182) nor at 12 months (n 197) was sweetness exposure associated with sweetness liking. While sweetness liking at 3 months was unrelated to liking at 6 months, the latter predicted sweetness liking at 12 months. These findings demonstrate no association between sweetness exposure at 3 to 12 months and liking at 6 and 12 months despite a sharp increase in sweetness exposure in that period. However, sweetness liking at 6 and 12 months was positively associated.

The Lack of Dopamine Transporter Is Associated With Conditional Associative Learning Impairments and Striatal Proteomic Changes.

Dopamine (DA) is critically involved in different functions of the central nervous system (CNS) including control of voluntary movement, affect, reward, sleep, and cognition. One of the key components of DA neurotransmission is DA reuptake by the DA transporter (DAT), ensuring rapid clearance of DA from the synaptic cleft. Thus, lack of DAT leads to persistent high extracellular DA levels. While there is strong evidence for a role of striatal dopaminergic activity in learning and memory processes, little is known about the contribution of DAT deficiency to conditional learning impairments and underlying molecular processes. DAT-knockout (DAT-KO) rats were tested in a set of behavioral experiments evaluating conditional associative learning, which requires unaltered striatal function. In parallel, a large-scale proteomic analysis of the striatum was performed to identify molecular factors probably underlying behavioral patterns. DAT-KO rats were incapable to acquire a new operant skill in Pavlovian/instrumental autoshaping, although the conditional stimulus-unconditional stimulus (CS-US) association seems to be unaffected. These findings suggest that DAT directly or indirectly contributes to the reduction of transference of incentive salience from the reward to the CS. We propose that specific impairment of conditional learning might be caused by molecular adaptations to the hyperdopaminergic state, presumably by dopamine receptor 1 (DRD1) hypofunction, as proposed by proteomic analysis. Whether DRD1 downregulation can cause cognitive deficits in the hyperdopaminergic state is the subject of discussion, and further studies are needed to answer this question. This study may be useful for the interpretation of previous and the design of future studies in the dopamine field.

Onset and burden of lower limb lymphedema after radical prostatectomy: a cross-sectional study.

OBJECTIVES: To explore men's onset and burden of lower limb lymphedema (LLL) after radical prostatectomy (RP) with pelvic lymph node dissection (PLND). PATIENTS AND METHODS: A cross-sectional survey-based study was conducted nation-wide and web-based in Germany. Part 1 included 15 multidisciplinary compiled questions with three questions from the Short Form 12 Health Survey (SF-12) and the WHO activity recommendation and part 2 included the validated German Lymph-ICF-Questionnaire (Lymph-ICF-LL). Subgroup comparisons and simple regression analyses were used to identify factors associated with therapy and burden of LLL, followed by multiple regression analyses to explain variance in impairment in the patients' daily life. RESULTS: Fifty-four patients completed the survey. Median time of LLL-onset was reported with 2.0 (0.5-9.75) months after RP. Nineteen patients (35.2%) reported bilateral lymphedema, 28 (51.9%) the use of individually fitted compression stockings (CS), 25 (46.3%) of manual lymphatic drainage (LD), and 26 (48.1%) complete regression. The Lymph-ICF-LL revealed a higher total burden for patients with an active LLL compared to complete regression (total score: 25.5 vs. 11.9, p = 0.01) especially for "physical function" (28.3 vs. 12.9, p < 0.01) and "mental function" (26.2 vs. 6.7, p < 0.01). In multiple linear regression analysis, a higher BMI (ß = 0.28), lower subjective general health (ß = -0.48), and active lymphedema (ß = 0.28) were significant predictors of higher reported impairments in the Lymph-ICF-LL, accounting for 45.4% of variance. CONCLUSION: Men with LLL after RP with PLND report a significant burden in daily life. Basic therapy needs to be offered early. Postoperative onset of LLL is variable, which should be considered when assessing complications after RP.

Prevalence of RND efflux pump regulator variants associated with tigecycline resistance in carbapenem-resistant Acinetobacter baumannii from a worldwide survey.

OBJECTIVES: To determine the most common tigecycline resistance mechanisms in carbapenem-resistant Acinetobacter baumannii isolates obtained during the global Tigecycline Evaluation Surveillance Trial (TEST). METHODS: Tigecycline MICs were determined by broth microdilution. WGS was used to screen for the previously identified tigecycline resistance mechanisms, as well as mutations in resistance-nodulation-cell division (RND)-type efflux pump regulators. RESULTS: From a total 313 isolates, 113 genetically unique tigecycline-resistant isolates were analysed. The most frequent and worldwide distributed mechanism associated with tigecycline resistance was disruption of adeN, which encodes the repressor of the RND efflux pump AdeIJK, either by IS elements or nucleotide deletions causing premature stop codons. However, mutations leading to amino acid substitutions and disruption by IS elements within the two-component regulatory system adeRS, which regulates expression of the AdeABC efflux pump, correlate with higher tigecycline MICs, but these were found less frequently and were mainly restricted to Southern European countries. Furthermore, an altered version of tviB was identified in several tigecycline-resistant isolates that did not have putative resistance mutations within RND-type regulators. The resistance determinants tet(A) and tet(X), as well as resistance mutations in putative resistance determinants trm, plsC, rrf, msbA and genes encoding 30S ribosomal proteins, were not identified in any isolate. CONCLUSIONS: The most prevalent tigecycline resistance mechanisms were caused by alterations in the regulators of RND-type efflux pumps. These data provide the basis for further characterization of regulator alterations and their contribution to increased efflux and tigecycline resistance, and also should be taken into account in drug discovery programmes to overcome the contribution of efflux pumps.

Reduced chromatin binding of MYC is a key effect of HDAC inhibition in MYC amplified medulloblastoma.

BACKGROUND: The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. METHODS: Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. RESULTS: HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. CONCLUSIONS: Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC's transactivating and transrepressing functions.

In vitro activity of sulbactam/durlobactam against global isolates of carbapenem-resistant Acinetobacter baumannii.

OBJECTIVES: To evaluate the activity of the novel broad-spectrum serine ß-lactamase inhibitor durlobactam (ETX2514) combined with sulbactam against global isolates of carbapenem-resistant Acinetobacter baumannii with defined carbapenem resistance mechanisms compared with reference antimicrobials with known activity against Acinetobacter spp. METHODS: The susceptibility of 246 carbapenem-resistant non-duplicate A. baumannii isolates to sulbactam/durlobactam, amikacin, colistin, imipenem/sulbactam/durlobactam, imipenem, meropenem, minocycline and sulbactam was tested using broth microdilution. Isolates were obtained from various body sites from patients in 37 countries and from six world regions between 2012 and 2016. Identification of carbapenem resistance mechanisms and assignment to A. baumannii clonal lineages was based on WGS. RESULTS: Sulbactam/durlobactam showed excellent activity comparable to colistin but superior to amikacin, minocycline and sulbactam. The sulbactam/durlobactam MIC50/90 values were 1/4 and 2/4 mg/L and the colistin MIC50/90 values were 0.5 and 1 mg/L, respectively. Comparatively, amikacin, minocycline and sulbactam MIC50/90 values were 256/≥512, 2/16 and 16/64 mg/L, respectively. CONCLUSIONS: Sulbactam/durlobactam had excellent in vitro potency against A. baumannii isolates, including those that were resistant to imipenem/meropenem, amikacin, minocycline and colistin, compared with other compounds. Sulbactam/durlobactam has the potential to become a useful addition to the limited armamentarium of drugs that can be used to treat this problem pathogen.

Development of a Reporter System to Explore MMEJ in the Context of Replacing Large Genomic Fragments.

Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by mhs.

Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion.

Pleiotropic corepressors Sin3 and Ssn6 interact with repressor Opi1 and negatively regulate transcription of genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

Repressor protein Opi1 is required to negatively regulate yeast structural genes of phospholipid biosynthesis in the presence of precursor molecules inositol and choline (IC). Opi1 interacts with the paired amphipathic helix 1 (PAH1) of pleiotropic corepressor Sin3, leading to recruitment of histone deacetylases (HDACs). Mutational analysis of the Opi1-Sin3 interaction domain (OSID) revealed that hydrophobic OSID residues L56, V59 and V67 of Opi1 are indispensable for gene repression. Our results also suggested that repression is not executed entirely via Sin3. Indeed, we could show that OSID contacts a second pleiotropic corepressor, Ssn6 (=Cyc8), which together with Tup1 is also able to recruit HDACs. Interestingly, mutations sin3 and ssn6 turned out as synthetically lethal. Our analysis further revealed that OSID not only binds to PAH1 but also interacts with tetratricopeptide repeats (TPR) of Ssn6. This interaction could no longer be observed with Opi1 OSID variants. To trigger gene repression, Opi1 must also interact with activator Ino2, using its activator interaction domain (AID). AID contains a hydrophobic structural motif reminiscent of a leucine zipper. Our mutational analysis of selected positions indeed confirmed that residues L333, L340, V343, V350, L354 and V361 are necessary for repression of Opi1 target genes.

Immunoadsorption in dilated cardiomyopathy: long-term reduction of cardiodepressant antibodies.

BACKGROUND: Disturbances of humoral immunity have been described in dilated cardiomyopathy (DCM), and a number of antibodies against cardiac cell proteins have been identified. Previous studies showed that immunoadsorption therapy with subsequent IgG substitution (IA/IgG) enhances cardiac function, and that removal of cardiodepressant antibodies may represent one essential mechanism of this therapy. The long-term effect of IA/IgG on the level of cardiodepressant antibodies remains to be elucidated. METHODS: A total of 17 patients with DCM were observed up to 12 months after IA/IgG. Echocardiographic measurements were performed at baseline, 3, 6 and 12 months after therapy. Cardiodepressant antibodies were detected by incubation of rat cardiomyocytes with purified patients' IgG and recording of contractility and Ca(2+) ratio. RESULTS: In contrast to patients without cardiodepressant antibodies before IA/IgG, patients with negative inotropic antibodies showed an improvement of left ventricular ejection fraction (LVEF) from 33.8 +/- 1.7% to 44.7 +/- 2.7%; 44.5 +/- 2.3% and 51.8 +/- 1.7% after 3, 6 and 12 months (P < 0.001 vs. baseline, P < 0.05 vs. LVEF of non-cardiodepressant group). Immediately after IA/IgG therapy, no cardiodepressant effects of patients' IgG on isolated cardiomyocytes were detectable, and this effect remained diminished until 6 months after IA/IgG (P < 0.001 for contractility and Ca(2+) ratio). Compared with the levels after 3 and 6 months, cardiodepressant antibodies reoccured after 12 months (P = 0.067 for contractility, P < 0.05 for Ca(2+) ratio vs. 6 months after IA/IgG). However, the negative inotropic reaction is still diminished compared with the reaction before IA/IgG. CONCLUSION: IA/IgG therapy induces long-term reduction of negative inotropic antibodies. After 12 months, however, re-increase of negative inotropic antibodies cannot be excluded.