Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Factors associated with misreporting in performance-based financing in Burkina Faso: Implications for risk-based verification.

Performance-based financing (PBF) has been piloted in many low- and middle-income countries (LMICs) as a strategy to improve access to and quality of health services. As a key component of PBF, quantity verification is carried out to ensure that reported data matches the actual number of services provided. However, cost concerns have led to a call for risk-based verification. Existing evidence suggests misreporting is associated with factors such as complexity of indicators, high service volume, and accepted error margin. In contrast, evidence on the association of key facility characteristics with misreporting in PBF is scarce. We contributed to filling this gap in knowledge by combining administrative data from a large-scale pilot PBF program in Burkina Faso with data from a health facility assessment in the context of an impact evaluation of the intervention. Our results showed the coexistence of both overreporting and underreporting and that misreporting varied by service indicator and health district. We also found that the number of clinical staff at the facility, the population size in the facility catchment area, and the distance between the facility and the district administration were associated with the probability of misreporting. We recommend further research of these factors in the move towards risk-based verification. In addition, given that our analysis identified relevant associations, but could not explain them, we recommend further qualitative inquiry into verification processes.

Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum.

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.

Nightly Biting Cycles of Anopheles Species in Rural Northwestern Burkina Faso.

The biting behavior of anophelines is an important determinant of malaria transmission. Understanding the local vector host-seeking behavior, its outdoor/ indoor biting preference, and nocturnal biting periods is essential for effectively applying and improving vector control methods, such as Long Lasting Insecticidal Nets (LLINs) and personal protective measures. To better understand the biting and host-seeking patterns of Anopheles mosquitoes in Northwestern Burkina Faso, we performed biweekly Human Landing Catches (HLC) in six villages during the period of highest mosquito abundance and malaria transmission. We applied a negative binomial regression framework to statistically analyze the host-seeking activities of Anopheles species and test for differences across hours, months, and villages, as well as for differences between indoor and outdoor capture points. Anopheles gambiae s.l. was identified as the main malaria vector in this region, representing about 90% of the total anopheline population. Biting activity was significantly different across hours and showed a peaked plateau between 2000 and 0200 hours. Differences in the pattern of biting cycles were observed between the early and late rainy season. This study shows that anopheline biting activity in Northwest Burkina Faso is high throughout the night, at indoor and outdoor posts alike. Consequently, bed nets alone may not provide sufficient protection against early biting anophelines and should be complemented with additional strategies such as indoor residual spraying (IRS) and larval source management (LSM) to meet the WHO's ambitious goals that are reflected in the global technical malaria strategy for 2030.

Efficacy of peginterferon plus ribavirin in patients receiving opioid substitution therapy : Final results of the Austrian PegHope study.

BACKGROUND: Patients with a history of intravenous drug abuse included in an official opioid substitution program represent an important subgroup of patients with chronic hepatitis C. The objective of this study was to assess the efficacy of and adherence to treatment with peginterferon and ribavirin in Austrian patients on stable opioid substitution therapy (OST). METHODS: This prospective, multicenter, observational, non-interventional trial (clinicaltrials.gov identifier, NCT01416610) included treatment-naïve patients with chronic hepatitis C on OST. Treatment consisted of peginterferon alpha-2a (PEGASYS®, 180 µg/week) plus ribavirin (COPEGUS®, 1000/1200 mg/day in genotypes (GT) 1/4 and 800 mg/day in GT 2/3) for 24-72 weeks, according to GT and viral response. RESULTS: The intention-to-treat (ITT) population comprised 88 patients. Mean duration of therapy was 6.0 ± 2.8 months. Treatment was discontinued earlier than planned in 34 out of 88 patients (39%), mainly because of poor adherence or side effects of treatment. At the end of treatment 65/88 patients (74%) were PCR negative. During follow-up, 5 patients relapsed. Only 44/88 patients (50%) could be evaluated 24 weeks after the end of treatment. Sustained virologic response 24 weeks after end of therapy (SVR24) was documented in 39/88 patients (44%). If only patients were considered who finished treatment as planned and for whom results at follow-up week 24 were available, the SVR24 rate was 89% (32/36). CONCLUSION: Despite favorable prognostic factors, such as young age and a high proportion of GT3, SVR rates were low in this cohort of patients receiving OST, the main reason being poor adherence; however, in those patients completing treatment, the SVR rate was high.

Efficacy of ledipasvir/sofosbuvir plus ribavirin for 12 weeks in patients with chronic hepatitis C genotype 3 and compensated liver disease.

INTRODUCTION: In the era of direct-acting antivirals, hepatitis C virus (HCV) genotype (GT) 3 remains as the most difficult-to-treat HCV-GT. Currently, data on the efficacy of ledipasvir/sofosbuvir plus ribavirin (SOF/LDV+RBV) in GT3-infected patients are limited. We investigated the efficacy of this regimen in a real-life cohort from Austria. PATIENTS AND METHODS: A total of 55 patients with HCV-GT3 and compensated liver disease (20% treatment-experienced, 33% with cirrhosis, 7% with HIV coinfection) from four Austrian hepatitis centers received treatment with SOF/LDV+RBV for 12 weeks. The primary endpoint was sustained virological response 12 weeks after end of therapy (SVR12). RESULTS: In the modified intention-to-treat analysis - excluding patients lost to follow-up - the overall SVR12 rate was 94% (95% confidence interval: 84-99%). In treatment-naive and treatment-experienced patients, SVR12 rates were 95 and 89%, respectively. SVR12 rate was 91% in patients without cirrhosis and 100% in patients with cirrhosis. There were no serious adverse events. Viral sequencing did not show the presence of any resistance-associated substitutions in any of the three relapsed patients. CONCLUSION: Despite a very weak antiviral activity of ledipasvir against HCV-GT3 in vitro, a 12-week course of SOF/LDV+RBV was highly effective, with a 94% SVR12 rate in our cohort of compensated HCV-GT3-infected patients. Thus, if pangenotypic NS5A inhibitors are not available or not reimbursed by insurances, SOF/LDV+RBV seems to be an effective alternative in patients with HCV-GT3 infection.

Filamin A promotes efficient migration and phagocytosis of neutrophil-like HL-60 cells.

The primary defense machinery to combat inflammation involves neutrophil granulocytes which in order to execute their functions rely on the efficiency of different cellular mechanisms including adhesion, spreading, migration in different environments, and phagocytosis. These functions require an accurately regulated actin network as well as the activation and adjustment of various signaling pathways. Mammalian filamins (FLNs) comprise three highly homologous large actin-binding proteins that are obvious candidates to control these processes as FLNs have been described to play a role in migration, spreading and adhesion in a variety of different cell types. The present study analyzed the role of filamin A (FLNa) in human neutrophil-like HL-60 cells. We found a strong enrichment of FLNa at the uropod of migrating neutrophils, and show that deficiency of FLNa caused a decrease in speed of migration both in 2D and 3D that is accompanied by a reduced activation of myosin-II. In addition, we show that FLNa plays a role in neutrophil phagocytosis. We also identified a hitherto unknown interaction of FLNa with coronin 1A that is mediated by FLNa repeats 9-18. FLNa deficiency had no or only minor effects on cell adhesion and spreading. In summary, deficiency of FLNa in human neutrophil-like HL-60 cells resulted in a surprisingly subtle phenotype. Our data indicate that FLNa is not essential for the regulation of mechanical properties during migration, but contributes to motility in a modulatory manner probably through its action at the uropod.

Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4.

Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.

EB1 contributes to proper front-to-back polarity in neutrophil-like HL-60 cells.

Directed migration of leukocytes towards a chemotactic source is largely dependent on coordinated actin cytoskeleton functions that provide the driving forces at the cell front and enable contractility at the rear. In contrast to the force-generating properties of the actin cytoskeleton, the microtubule network assumes a regulatory function in balancing front-to-back polarity. In migrating neutrophils, microtubules are mostly concentrated at the cell rear, and previously published work suggested that microtubules are stabilized and kept in place by a mechanism involving Cdc42, WASP, CD11b, and the end-binding protein 1 (EB1). EB1, as a microtubule plus-end tracking protein (+TIP), is a potential candidate to bridge the gap between microtubule and actomyosin dynamics. After knockdown of EB1 in neutrophil-like HL-60 cells, both directionality and straightness of migration while moving through 3D collagen gels are impaired. An increased number of lateral protrusions are observed in EB1-knockdown cells, indicating an inability to balance cell polarity in the absence of EB1. Moreover, in EB1-deficient cells, substrate adhesion on fibrinogen-coated surfaces is significantly reduced. EB1-knockdown cells show significant changes in levels of GEF-H1, a microtubule-associated guanine nucleotide exchange factor that links microtubule integrity to RhoA-dependent regulation of the actin cytoskeleton, suggesting that GEF-H1 might constitute one element of the microtubule-actin crosstalk in migrating leukocytes.

Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum.

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

Routine implementation costs of larviciding with Bacillus thuringiensis israelensis against malaria vectors in a district in rural Burkina Faso.

BACKGROUND: The key tools in malaria control are early diagnosis and treatment of cases as well as vector control. Current strategies for malaria vector control in sub-Saharan Africa are largely based on long-lasting insecticide-treated nets (LLINs) and to a much smaller extent on indoor residual spraying (IRS). An additional tool in the fight against malaria vectors, larval source management (LSM), has not been used in sub-Saharan Africa on a wider scale since the abandonment of environmental spraying of DDT. Increasing concerns about limitations of LLINs and IRS and encouraging results from large larvicide-based LSM trials make a strong case for using biological larviciding as a complementary tool to existing control measures. Arguments that are often quoted against such a combined approach are the alleged high implementation costs of LSM. This study makes the first step to test this argument. The implementation costs of larval source management based on Bacillus thuringiensis israelensis (Bti) (strain AM65-52) spraying under different implementation scenarios were analysed in a rural health district in Burkina Faso. METHODS: The analysis draws on detailed cost data gathered during a large-scale LSM intervention between 2013 and 2015. All 127 villages in the study setup were assigned to two treatment arms and one control group. Treatment either implied exhaustive spraying of all available water collections or targeted spraying of the 50 % most productive larval sources via remote-sensing derived and entomologically validated risk maps. Based on the cost reports from both intervention arms, the per capita programme costs were calculated under the assumption of covering the whole district with either intervention scenario. Cost calculations have been generalized by providing an adaptable cost formula. In addition, this study assesses the sensitivity of per capita programme costs with respect to changes in the underlying cost components. RESULTS: The average annual per capita costs of exhaustive larviciding with Bti during the main malaria transmission period (June-October) in the Nouna health district were calculated to be US$ 1.05. When targeted spraying of the 50 % most productive larval sources is used instead, average annual per capita costs decrease by 27 % to US$ 0.77. Additionally, a high sensitivity of per capita programme costs against changes in total surface of potential larval sources and the number of spraying repetitions was found. DISCUSSION: The per capita costs for larval source management interventions with Bti are roughly a third of the annual per capita expenditures for anti-malarial drugs and those for LLINs in Burkina Faso which are US$ 3.80 and 3.00, respectively. The average LSM costs compare to those of IRS and LLINs for sub-Saharan Africa. The authors argue that in such a setting LSM based on Bti spraying is within the range of affordable anti-malarial strategies and, consequently, should deserve more attention in practice. Future research includes a cost-benefit calculation, based on entomological and epidemiological data collected during the research project.

Assembly of methylated KDM1A and CHD1 drives androgen receptor-dependent transcription and translocation.

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments.

Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro. In the present study, we set out to revisit the role of ddFLN in cell migration. For this purpose, we examined migration of wild-type, ddFLN-null and ddFLN-overexpressing cells under different conditions. In addition to cyclic-AMP chemotaxis assays using micropipettes, we explored cell migration under more confined conditions: an under-agarose 2D assay and a 3D assay employing a collagen matrix that was adapted from assays for leukocytes. Using 3D migration conditions, cells deficient in ddFLN displayed only a minor impairment of motility, similar to the results obtained for migration in 2D. However, cells overexpressing ddFLN showed a remarkable decrease in the speed of migration in particular in 3D environments. We suggest that these results are in line with an increased stiffening of the cortex due to the crosslinking activity of overexpressed ddFLN. Our conclusion is that the absolute level of ddFLN is critical for efficient migration. Furthermore, our results show that under conditions of increased mechanical stress, Dictyostelium cells, like leukocytes, switch to a bleb-based mode of movement.

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement.

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting.

Stromal cell-derived factor 2 is critical for Hsp90-dependent eNOS activation.

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway.

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction.