Polyphosphate co-localizes with factor XII on plateletbound fibrin and augments its plasminogen activator activity

This work was supported by grants FS/11/2/28579 (N.J.M. & A.S.L) from the British Heart Foundation and by the University of Aberdeen Development Trust (J.L.M. & N.J.M.). P.Y.K is supported by an Early Career Award and New Investigator Fund from the Hamilton Health Sciences. We are grateful to the following students for contributions to the project, Natasha Walker & Thomas Nolan. We also thank both the Microscopy and Histology Core Facility and the Iain Fraser Cytometry Centre at the University of Aberdeen for excellent advice and use of the facilities. We also thank Dr Jeffrey Weitz from McMaster University, Canada for the kind gift of HRG.Peer reviewedPostprin

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

<p>Abstract</p> <p>Background</p> <p>In male <it>Drosophila melanogaster</it>, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.</p> <p>Results</p> <p>MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in <it>Drosophila</it>. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity <it>in vitro </it>and UAS-DsRed activation in <it>Drosophila</it>. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-<it>lacZ </it>and UAS-<it>arm-lacZ </it>reporter genes. The latter utilizes the constitutive promoter from the <it>arm </it>gene to drive <it>lacZ </it>expression. In contrast to the strong induction of UAS-DsRed expression, UAS-<it>arm-lacZ </it>expression increased by about 2-fold in both sexes.</p> <p>Conclusions</p> <p>Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-<it>DsRed </it>but not UAS-<it>arm-lacZ </it>genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.</p

Progeny counter mechanism in malaria parasites is linked to extracellular resources.

Malaria is caused by the rapid proliferation of Plasmodium parasites in patients and disease severity correlates with the number of infected red blood cells in circulation. Parasite multiplication within red blood cells is called schizogony and occurs through an atypical multinucleated cell division mode. The mechanisms regulating the number of daughter cells produced by a single progenitor are poorly understood. We investigated underlying regulatory principles by quantifying nuclear multiplication dynamics in Plasmodium falciparum and knowlesi using super-resolution time-lapse microscopy. This confirmed that the number of daughter cells was consistent with a model in which a counter mechanism regulates multiplication yet incompatible with a timer mechanism. P. falciparum cell volume at the start of nuclear division correlated with the final number of daughter cells. As schizogony progressed, the nucleocytoplasmic volume ratio, which has been found to be constant in all eukaryotes characterized so far, increased significantly, possibly to accommodate the exponentially multiplying nuclei. Depleting nutrients by dilution of culture medium caused parasites to produce fewer merozoites and reduced proliferation but did not affect cell volume or total nuclear volume at the end of schizogony. Our findings suggest that the counter mechanism implicated in malaria parasite proliferation integrates extracellular resource status to modify progeny number during blood stage infection

An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin levels. Disruption of tubulin homeostasis caused excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed mitotic spindle extension but did not significantly interfere with DNA replication. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome

Combined Focused Next-Generation Sequencing Assays to Guide Precision Oncology in Solid Tumors: A Retrospective Analysis from an Institutional Molecular Tumor Board

Background: Increasing knowledge of cancer biology and an expanding spectrum of molecularly targeted therapies provide the basis for precision oncology. Despite extensive gene diagnostics, previous reports indicate that less than 10% of patients benefit from this concept. Methods: We retrospectively analyzed all patients referred to our center&rsquo;s Molecular Tumor Board (MTB) from 2018 to 2021. Molecular testing by next-generation sequencing (NGS) included a 67-gene panel for the detection of short-sequence variants and copy-number alterations, a 53- or 137-gene fusion panel and an ultra-low-coverage whole-genome sequencing for the detection of additional copy-number alterations outside the panel&rsquo;s target regions. Immunohistochemistry for microsatellite instability and PD-L1 expression complemented NGS. Results: A total of 109 patients were referred to the MTB. In all, 78 patients received therapeutic proposals (70 based on NGS) and 33 were treated accordingly. Evaluable patients treated with MTB-recommended therapy (n = 30) had significantly longer progression-free survival than patients treated with other therapies (n = 17) (4.3 vs. 1.9 months, p = 0.0094). Seven patients treated with off-label regimens experienced major clinical benefits. Conclusion: The combined focused sequencing assays detected targetable alterations in the majority of patients. Patient benefits appeared to lie in the same range as with large-scale sequencing approaches

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2

Purpose!#!Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection.!##!Methods!#!This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use.!##!Results!#!The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2-87.5%). Specificity was 99.3% (CI 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings.!##!Conclusion!#!The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.!##!Trial registration number and registration date!#!DRKS00021220 and 01.04.2020