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Institute
Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.
Osteocalcin, Azan and Toluidine blue staining in fibrous dysplasia and ossifying fibroma of the jaws
(2018)
Background: Fibrous dysplasia (FD) and ossifying fibroma (OF) are fibro-osseous lesions (FOLs) having several overlaps that may make final diagnosis difficult by hematoxylin and eosin (H/E) alone.
Aim: This study seeks to detect any association between Azan and Toluidine blue staining as compared with osteocalcin in FD and OF diagnosis.
Methods:Forty formalin fixed paraffin embedded (FFPE) blocks of FD and OF were prepared for Azan, Toluidine blue and osteocalcin staining. Brown staining of calcified structures was considered as positive for osteocalcin. Scoring for Azan and Toluidine blue was evaluated based on intensity and localization. Level of agreement of original and revised diagnosis was determined.
Results: Six (40%) of 15 FD were corroborated by osteocalcin. Eight cases initially diagnosed as OF were revised to FD. There were 25 OF according to H/E, and 17 (68%) were validated by osteocalcin. Measure of agreement between histology and immunohistochemistry was 0.081; p = .608. Eleven (42.3%) OF expressed strong toluidine blue staining of the intervening fibrous connective tissue stroma while only 2 (14.2%) FD showed similar staining, this difference was statistically significant [p = .001].
Conclusions: Histomorphometric analysis with Toluidine blue may reduce diagnostic errors of OF and FD.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
After entorhinal deafferentiation of the hippocampal dentate gyrus a reinnervation of the denervated neurons by axon collaterals can be observed. This process takes place in a matter of weeks. However, the overall functional effect on the hippocampal network is still unclear.
In an effort to investigate this effect of axonal sprouting on the neuronal network of the dentate gyrus we compared the electrophysiological response of the dentate gyrus after electric stimulation in wild-type mice (WT mice) with a normal post-lesion sprouting, with genetically modified mice with an overexpression of the growth-protein CAP23 (cytoskeleton-associated protein 23). CAP23 overexpressing mice (CAP23tg mice) are known to have an enhanced axonal growth and sprouting after lesion.
The mice (both the WT as well as the CAP23tg mice) were deeply anesthetized and a lesion of the perforant path was induced stereotactically with a wire knife. After that the mice were permitted to survive for 4-6 weeks for partial reinnervation of the dentate gyrus before they were again operated and evoked potentials were measured (extracellular recordings of evoked potentials in the dentate gyrus). Non-lesioned litter-mate mice were taken as reference. The sprouting and the correct position of the electrodes was confirmed histologically.
For electrophysiological investigation we assessed laminar profiles and calculated a current-source density (CSD). In lesioned CAP23tg mice compared to lesioned WT mice this CSD-analysis revealed a significant enhancement of the current sink in the area of deafferentiation (outer molecular layer) and a significant excitation in the granule-cell layer.
Our results show that axonal sprouting seems to enhance the excitability of granule-cells. Thus, even if an enhanced axonal sprouting might accelerate the reinnervation of denervated dendrites after lesion, but it also leads to posttraumatic hyperexcitability of the neuronal network. In a therapeutic approach of fascilitating axonal sprouting this hyperexcitability has to be taken into consideration.
MAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary disfunction and by defects in function, activation and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases.
Purpose: To report a case of autoimmune keratitis in a patient with mycobacterium tuberculosis (MBT).
Methods: An 84-year-old male with pulmonary tuberculosis (TB) was admitted with chronic, non-healing bilateral ulcerations of the inferior peripheral cornea associated with stromal and subconjunctival nodules.
Results: Clinical examination revealed circumscribed peripheral corneal ulceration with whitish nodules in adjacent stromal and subconjunctival tissue. Microbiological cultures of the corneal tissue were negative for MBT and other microbial pathogens; however, enzyme-linked immunosorbent assay (ELISA) of blood and corneal samples showed significantly elevated levels of IgM and IgA against MBT. In addition to systemic anti-tuberculosis therapy, the patient was treated topically with Polyspectran® eye drops, Dexamethasone eye drops, and Bepanthen® ointment, for 2 weeks. Both eyes showed dramatic improvement after 2 weeks.
Conclusion: The present report demonstrates that MBT is able to initiate delayed autoimmune response within the corneal tissue during an intensive phase of anti-tuberculosis treatment.
Background: MDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.
Methods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.
Results: All MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.
Conclusion: We introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.
Objectives Omeprazole was shown to improve the anti-cancer effect of the nucleoside-analogue 5-fluorouracil. Here, we investigated the effects of omeprazole on the activities of the antiviral nucleoside analogues ribavirin and acyclovir.
Methods West Nile virus-infected Vero cells and influenza A H1N1-infected MDCK cells were treated with omeprazole and/ or ribavirin. Herpes simplex virus 1 (HSV-1)- or HSV-2-infected Vero or HaCat cells were treated with omeprazole and/ or acyclovir. Antiviral effects were determined by examination of cytopathogenic effects (CPE), immune staining, and virus yield assay. Cell viability was investigated by MTT assay.
Results Omeprazole concentrations up to 80μg/mL did not affect the antiviral effects of ribavirin. In contrast, omeprazole increased the acyclovir-mediated effects on HSV-1- and HSV-2-induced CPE formation in a dose-dependent manner in Vero and HaCat cells. Addition of omeprazole 80μg/mL resulted in a 10.8-fold reduction of the acyclovir concentration that reduces CPE formation by 50% (IC50) in HSV-1-infected Vero cells and in a 47.7-fold acyclovir IC50 reduction in HSV-1-infected HaCat cells. In HSV-2-infected cells, omeprazole reduced the acyclovir IC50 by 7.3-fold (Vero cells) or by 12.9-fold (HaCat cells). Omeprazole also enhanced the acyclovir-mediated effects on viral antigen expression and virus replication in HSV-1- and HSV-2-infected cells. In HSV-1-infected HaCat cells, omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 1μg/mL by 1.6×105-fold. In HSV-2-infected HaCat cells omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 2μg/mL by 9.2×103-fold. The investigated drug concentrations did not affect cell viability, neither alone nor in combination.
Conclusions Omeprazole increases the anti-HSV activity of acyclovir. As clinically well-established and tolerated drug, it is a candidate drug for antiviral therapies in combination with acyclovir.
Prenatal and postnatal experiences associated with epigenetic changes in the adult mouse brain
(2018)
To analyze the influences of early-life history on the brain epigenome, the offspring of mouse dams kept in an enriched or standard environment were exposed postnatally to enriched, standard, or adverse conditions. The methylation patterns of 7 candidate genes (9 loci) involved in developmental programming of stress vulnerability/resilience and psychiatric disease were analyzed in 6 brain regions of adult male and female mice. Exposure to an enriched prenatal environment was associated with widespread epigenetic changes (all of small effect size), affecting 29 of 324 (9%) gene/region-specific methylation patterns. The effects of either adverse or enriched postnatal conditions were tested separately in the two prenatal cohorts. Significant changes were observed in 2 of 324 (0.6%) loci in offspring of dams in a standard environment and 6 of 324 (1.9%) loci in animals that were exposed prenatally to an enriched environment. Prenatal life experiences appear to have a bigger effect on the adult brain epigenome than postnatal experiences. Positive prenatal life experiences may increase epigenetic plasticity of the brain later in life. All observed between-group differences were sex-specific, consistent with largely different developmental trajectories of the male and female brain. Multiple changes of small effect size are consistent with a multifactorial model of developmental programming of adult behavior and disease susceptibility.
Introduction: Ferroptosis has recently been identified as a form of programmed cell death caused by an accumulation of lipid reactive oxygen species (ROS). However, little is yet known about the role in hepatocellular carcinoma (HCC) and its signalling mechanism as well the modulation of ROS.
Material and methods: Human HCC cell lines were treated with different concentrations of ROS modulators (Auranofin, Erastin, BSO). Cell death was determined by analysis of PI-stained nuclei using flow cytometry. ROS production and lipid peroxidation were analysed at early time points before cell death starts. For mechanistic studies we performed Western Blot and a Proteome array. Different inhibitors of cell death target proteins, ROS-scavengers as well as lipoxygenase inhibitors were used. To investigate the functional relevance of NAPDH oxidases (NOX) 1 and 4 for ROS modulation and ferroptosis we genetically silenced its genes using three distinct siRNAs and we used the NOX1/4-inhibitor GKT137831.
Results and discussions: Compared to the single treatment, Auranofin/BSO-cotreatment as well as Erastin/BSO-cotreatment acted in concert to trigger cell death and to reduce cell viability of HCC cells in a dose- and time-dependent manner. Furthermore, both cotreatments induce ROS production, lipid peroxidation and ferroptotic cell death, which could be inhibited by the use of Ferrostatin-1 (inhibitor of lipid peroxidation) and Liproxstatin-1 (specific inhibitor of ferroptosis). The broad-range caspase inhibitor zVAD.fmk failed to rescue cells from Auranofin/BSO- or Erastin/BSO-cotreatment induced cell death. No activation of caspases-3 could be seen in the proteome profiler apoptosis assay. Importantly, the selective lipoxygenase (LOX) inhibitor Baicalain and the pan-LOX inhibitor NDGA protect HCC cells from Auranofin/BSO- and Erastin/BSO-cotreatment stimulated lipid peroxidation, ROS generation and cell death, indication that the induction of ferroptosis may bypass apoptosis resistance of HCC cells. Mechanistic studies showed that Auranofin/BSO-cotreatment decreased TrxR-activity, led to Nrf2 accumulation and promoted the activation of HO-1. In contrast, NOX 1 and 4 were involved in Erastin/BSO-mediated cell death and the use of the NOX1/4-inhibitor GKT137831 rescued HCC cells from the Erastin/BSO-induced cell death.
Conclusion: By providing new insights into the molecular regulation of ROS and ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in HCC cells.