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Objectives Supersaturating formulations hold great promise for delivery of poorly soluble active pharmaceutical ingredients (APIs). To profit from supersaturating formulations, precipitation is hindered with precipitation inhibitors (PIs), maintaining drug concentrations for as long as possible. This review provides a brief overview of supersaturation and precipitation, focusing on precipitation inhibition. Trial-and-error PI selection will be examined alongside established PI screening techniques. Primarily, however, this review will focus on recent advances that utilise advanced analytical techniques to increase mechanistic understanding of PI action and systematic PI selection.
Key Findings. Advances in mechanistic understanding have been made possible by the use of analytical tools such as spectroscopy, microscopy and mathematical and molecular modelling, which have been reviewed herein. Using these techniques, PI selection can instead be guided by molecular rationale. However, more work is required to see wide-spread application of such an approach for PI selection.
Conclusions PIs are becoming increasingly important in enabling formulations. Trial-and-error approaches have seen success thus far. However, it is essential to learn more about the mode of action of PIs if the most optimal formulations are to be realised. Robust analytical tools, and the knowledge of where and how they can be applied, will be essential in this endeavour.
The gradual heterogeneity of climatic factors pose varying selection pressures across geographic distances that leave signatures of clinal variation in the genome. Separating signatures of clinal adaptation from signatures of other evolutionary forces, such as demographic processes, genetic drift, and adaptation to non-clinal conditions of the immediate local environment is a major challenge. Here, we examine climate adaptation in five natural populations of the harlequin fly Chironomus riparius sampled along a climatic gradient across Europe. Our study integrates experimental data, individual genome resequencing, Pool-Seq data, and population genetic modelling. Common-garden experiments revealed a positive correlation of population growth rates corresponding to the population origin along the climate gradient, suggesting thermal adaptation on the phenotypic level. Based on a population genomic analysis, we derived empirical estimates of historical demography and migration. We used an FST outlier approach to infer positive selection across the climate gradient, in combination with an environmental association analysis. In total we identified 162 candidate genes as genomic basis of climate adaptation. Enriched functions among these candidate genes involved the apoptotic process and molecular response to heat, as well as functions identified in other studies of climate adaptation in other insects. Our results show that local climate conditions impose strong selection pressures and lead to genomic adaptation despite strong gene flow. Moreover, these results imply that selection to different climatic conditions seems to converge on a functional level, at least between different insect species.
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.
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.
In the last decades, energy modelling has supported energy planning by offering insights into the dynamics between energy access, resource use, and sustainable development. Especially in recent years, there has been an attempt to strengthen the science-policy interface and increase the involvement of society in energy planning processes. This has, both in the EU and worldwide, led to the development of open-source and transparent energy modelling practices.This paper describes the role of an open-source energy modelling tool in the energy planning process and highlights its importance for society. Specifically, it describes the existence and characteristics of the relationship between developing an open-source, freely available tool and its application, dissemination and use for policy making. Using the example of the Open Source energy Modelling System (OSeMOSYS), this work focuses on practices that were established within the community and that made the framework's development and application both relevant and scientifically grounded. Keywords: Energy system modelling tool, Open-source software, Model-based public policy, Software development practice, Outreach practice
Metacognition plays a pivotal role in human development. The ability to realize that we do not know something, or meta-ignorance, emerges after approximately five years of age. We aimed at identifying the brain systems that underlie the developmental emergence of this ability in a preschool sample.
Twenty-four children aged between five and six years answered questions under three conditions of a meta-ignorance task twice. In the critical partial knowledge condition, an experimenter first showed two toys to a child, then announced that she would place one of them in a box behind a screen, out of sight from the child. The experimenter then asked the child whether or not she knew which toy was in the box.
Children who answered correctly both times to the metacognitive question in the partial knowledge condition (n=9) showed greater cortical thickness in a cluster within left medial orbitofrontal cortex than children who did not (n=15). Further, seed-based functional connectivity analyses of the brain during resting state revealed that this region is functionally connected to the medial orbitofrontal gyrus, posterior cingulate gyrus and precuneus, and mid- and inferior temporal gyri.
This finding suggests that the default mode network, critically through its prefrontal regions, supports introspective processing. It leads to the emergence of metacognitive monitoring allowing children to explicitly report their own ignorance.
Multiplex families with a high prevalence of a psychiatric disorder are often examined to identify rare genetic variants with large effect sizes. In the present study, we analysed whether the risk for bipolar disorder (BD) in BD multiplex families is influenced by common genetic variants. Furthermore, we investigated whether this risk is conferred mainly by BD-specific risk variants or by variants also associated with the susceptibility to schizophrenia or major depression. In total, 395 individuals from 33 Andalusian BD multiplex families as well as 438 subjects from an independent, sporadic BD case-control cohort were analysed. Polygenic risk scores (PRS) for BD, schizophrenia, and major depression were calculated and compared between the cohorts. Both the familial BD cases and unaffected family members had significantly higher PRS for all three psychiatric disorders than the independent controls, suggesting a high baseline risk for several psychiatric disorders in the families. Moreover, familial BD cases showed significantly higher BD PRS than unaffected family members and sporadic BD cases. A plausible hypothesis is that, in multiplex families with a general increase in risk for psychiatric disease, BD development is attributable to a high burden of common variants that confer a specific risk for BD. The present analyses, therefore, demonstrated that common genetic risk variants for psychiatric disorders are likely to contribute to the high incidence of affective psychiatric disorders in the multiplex families. The PRS explained only part of the observed phenotypic variance and rare variants might have also contributed to disease development.
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.
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.