The influence of the 5 '-terminal nucleotide on AgoshRNA activity and biogenesis: importance of the polymerase III transcription initiation site

Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents. We verified that AgoshRNAs trigger site-specific cleavage of a complementary mRNA. Second, we document the importance of the identity of the 5΄-terminal nucleotide and its basepairing status for AgoshRNA activity. AgoshRNA activity is significantly reduced or even abrogated with C or U at the 5΄-terminal and is enhanced by introduction of a bottom mismatch and 5΄-terminal nucleotide A or G. The 5΄-terminal RNA nucleotide also represents the +1 position of the transcriptional promoter in the DNA, thus further complicating the analysis. Indeed, we report that +1 modification affects the transcriptional efficiency and accuracy of start site selection, with A or G as optimal nucleotide. These combined results allow us to propose general rules for the design and expression of potent AgoshRNA molecule

Stacking-Order-Dependent Excitonic Properties Reveal Interlayer Interactions in Bulk ReS₂

Rhenium disulfide, a member of the transition metal dichalcogenide family of semiconducting materials, is unique among 2D van der Waals materials due to its anisotropy and, albeit weak, interlayer interactions, confining excitons within single atomic layers and leading to monolayer-like excitonic properties even in bulk crystals. While recent work has established the existence of two stacking modes in bulk, AA and AB, the influence of the different interlayer coupling on the excitonic properties has been poorly explored. Here, we use polarization-dependent optical measurements to elucidate the nature of excitons in AA and AB-stacked rhenium disulfide to obtain insight into the effect of interlayer interactions. We combine polarization-dependent Raman with low-temperature photoluminescence and reflection spectroscopy to show that, while the similar polarization dependence of both stacking orders indicates similar excitonic alignments within the crystal planes, differences in peak width, position, and degree of anisotropy reveal a different degree of interlayer coupling. DFT calculations confirm the very similar band structure of the two stacking orders while revealing a change of the spin-split states at the top of the valence band to possibly underlie their different exciton binding energies. These results suggest that the excitonic properties are largely determined by in-plane interactions, however, strongly modified by the interlayer coupling. These modifications are stronger than those in other 2D semiconductors, making ReS2 an excellent platform for investigating stacking as a tuning parameter for 2D materials. Furthermore, the optical anisotropy makes this material an interesting candidate for polarization-sensitive applications such as photodetectors and polarimetry.</p

Pyruvate dehydrogenase kinase inhibitor dichloroacetate improves host control of Salmonella enterica serovar Typhimurium infection in human macrophages

Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium (Stm) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) - which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) - on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm, but interestingly not Mycobacterium tuberculosis, in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections.Immunogenetics and cellular immunology of bacterial infectious disease

Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials

Immunogenetics and cellular immunology of bacterial infectious disease

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.Immunogenetics and cellular immunology of bacterial infectious disease

Bioorthogonal correlative light-electron microscopy of mycobacterium tuberculosis in macrophages reveals the effect of antituberculosis drugs on subcellular bacterial distribution

Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution.Bio-organic SynthesisMedical Biochemistr

Breast cancer risk genes: association analysis in more than 113,000 women

BACKGROUNDGenetic testing for breast cancer susceptibility is widely used, but for many genes, evidence of an association with breast cancer is weak, underlying risk estimates are imprecise, and reliable subtype-specific risk estimates are lacking.METHODSWe used a panel of 34 putative susceptibility genes to perform sequencing on samples from 60,466 women with breast cancer and 53,461 controls. In separate analyses for protein-truncating variants and rare missense variants in these genes, we estimated odds ratios for breast cancer overall and tumor subtypes. We evaluated missense-variant associations according to domain and classification of pathogenicity.RESULTSProtein-truncating variants in 5 genes (ATM, BRCA1, BRCA2, CHEK2, and PALB2) were associated with a risk of breast cancer overall with a P value of less than 0.0001. Protein-truncating variants in 4 other genes (BARD1, RAD51C, RAD51D, and TP53) were associated with a risk of breast cancer overall with a P value of less than 0.05 and a Bayesian false-discovery probability of less than 0.05. For protein-truncating variants in 19 of the remaining 25 genes, the upper limit of the 95% confidence interval of the odds ratio for breast cancer overall was less than 2.0. For protein-truncating variants in ATM and CHEK2, odds ratios were higher for estrogen receptor (ER)-positive disease than for ER-negative disease; for protein-truncating variants in BARD1, BRCA1, BRCA2, PALB2, RAD51C, and RAD51D, odds ratios were higher for ER-negative disease than for ER-positive disease. Rare missense variants (in aggregate) in ATM, CHEK2, and TP53 were associated with a risk of breast cancer overall with a P value of less than 0.001. For BRCA1, BRCA2, and TP53, missense variants (in aggregate) that would be classified as pathogenic according to standard criteria were associated with a risk of breast cancer overall, with the risk being similar to that of protein-truncating variants.CONCLUSIONSThe results of this study define the genes that are most clinically useful for inclusion on panels for the prediction of breast cancer risk, as well as provide estimates of the risks associated with protein-truncating variants, to guide genetic counseling. (Funded by European Union Horizon 2020 programs and others.)Molecular tumour pathology - and tumour geneticsMTG1 - Moleculaire genetica en pathologie van borstkanke

The way the Mu’tazilites worked and studied in the 10th century

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