Engineering rFVIIa-loaded platelets; a novel approach to treating acute bleeding

Haemorrhage remains a leading cause of mortality around the world, resulting from both trauma and surgery. Current treatments for acute haemorrhage include blood products such as fresh frozen plasma, as well as platelet transfusion and recombinant clotting factors such as rFVIIa. However, the use of recombinant clotting factors is limited due to cost and adverse side effects resulting from increased thrombosis, such as increased rate of myocardial infarction and cerebrovascular accidents. Platelets are small anucleate cells that exist in very large quantities in our blood and, along with cross-linked fibrin from the coagulation cascade, are involved in forming a haemostatic plug upon exposure to damaged endothelium in a wound. They are metabolically active and undergo a process of platelet activation when stimulated by pro-thrombotic agonists such as thrombin resulting from the coagulation cascade. This then triggers a process whereby the contents of their granules are released locally to facilitate coagulation. This thesis explores the possibility of loading these platelet granules with recombinant clotting factors, in this case rFVIIa which has already been shown in clinical trials to result in a significant decrease in mortality from acute haemorrhage. The targeted delivery of this drug is explored, both through endocytosis and genetic engineering of megakaryocytes differentiated in vitro from induced pluripotent stem cells (iPSCs). These are evaluated with in vitro assays to model the process of clot formation, as well as an in vivo model of haemostasis to compare their efficacy to conventional treatments. Overall, this serves as a proof of concept of the engineering of platelet granules as a novel drug delivery system.Cambridge School of Clinical Medicine, the Rosetrees Trust and the Frank Edward Elmore Fun

The integrated stress response regulates BMP signalling through effects on translation.

BACKGROUND: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress-sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone morphogenetic proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. RESULTS: Here we show in Drosophila that GCN2 antagonises BMP signalling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impairs developmental BMP signalling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signalling. Moreover, loss of d4E-BP, a target of crc, augments BMP signalling in vitro and rescues tissue development in vivo. CONCLUSION: These results identify a novel mechanism by which the ISR modulates BMP signalling during development

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia

Combining long-term circuit mapping and network transcriptomics with SiR-N2c

An exciting frontier in circuit neuroscience lies at the intersection between neural network mapping and single-cell genomics. Monosynaptic rabies viruses provide a promising platform for the merger of circuit mapping methods with -omics approaches. However, three key limitations have hindered the extraction of physiologically meaningful gene expression profiles from rabies-mapped circuits: inherent viral cytotoxicity, high viral immunogenicity and virus-induced alteration of cellular transcriptional regulation. These factors alter the transcriptional and translational profiles of infected neurons and their neighboring cells. To overcome these limitations we applied a self-inactivating genomic modification to the less immunogenic rabies strain, CVS-N2c, to generate a self-inactivating CVS-N2c rabies virus (SiR-N2c). SiR-N2c not only eliminates undesired cytotoxic effects but also substantially reduces gene expression alterations in infected neurons and dampens the recruitment of innate and acquired immune responses, thus enabling open-ended interventions on neural networks and their genetic characterization using single-cell genomic approaches

Additional file 1: of The integrated stress response regulates BMP signalling through effects on translation

Figure S1. Modulation of the ISR delays developmental delay and causes wing venation defects. (A) Phenotypes of animals expressing ppp1r15 RNAi under the control of a panel of tissue-selective drivers. (B) Representative photomicrographs (5× objective) of w 1118 ;esgGAL4 (esg) and esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi) animals at 5 and 14 days after egg laying (AEL). Scale bar = 1 mm. (C) Representative photomicrographs of w1118;esgGAL4 (esg), esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi), esgGAL4 > UAS-gcn2 RNAi (esg > gcn2 RNAi) and esgGAL4 > UAS-gcn2;UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi;gcn2 RNAi) animals at 14 days AEL. (D) Quantification of indicated crosses at days 5 and 14 AEL. esgGAL4 > UAS-ppp1r15 RNAi (esg > ppp1r15 RNAi), esgGAL4 > UAS-dGCN2 RNAi (esg > gcn2 RNAi) and esgGAL4 > UAS-dPERK RNAi (esg > perk RNAi). n denotes number of animals counted. P values calculated using Χ2 statistic with Bonferroni correction for multiple comparisons. (E) Quantification of indicated crosses at days 5 and 14 AEL. enGAL4 > UAS-ppp1r15 RNAi (en > ppp1r15 RNAi), enGAL4 > UAS-gcn2 RNAi (en > gcn2 RNAi) and enGAL4 > UAS-perk RNAi (en > perk RNAi). n denotes number of animals counted. P values calculated using Χ 2 statistics with Bonferroni correction for multiple comparisons. (F) Representative photomicrographs of adult wings of the indicated genotypes. Scale bars = 250 μm. (PDF 1057 kb

Additional file 4: of The integrated stress response regulates BMP signalling through effects on translation

Tables S1–S15. Analysis of transcriptional data. Table S1. mRNAs induced in S2 cells expressing HA-crc for 3 h. Table S2. mRNAs induced in S2 cells expressing HA-crc for 6 h. Table S3. mRNAs repressed in S2 cells expressing HA-crc for 3 h. Table S4. mRNAs repressed in S2 cells expressing HA-crc for 6 h. Table S5. Gene Ontology (GO) term enrichment of mRNAs induced in S2 cells expressing HA-crc for 3 h. Table S6. GO term enrichment of mRNAs induced in S2 cells expressing HA-crc for 6 h. Table S7. GO term enrichment of mRNAs repressed in S2 cells expressing HA-crc for 3 h. Table S8. GO term enrichment of mRNAs repressed in S2 cells expressing HA-crc for 6 h. Table S9. mRNAs induced in S2 cells expressing dGCN2-CA-V5 for 6 h. Table S10. mRNAs induced in S2 cells expressing dGCN2-CA-V5 for 12 h. Table S11. mRNAs repressed in S2 cells expressing dGCN2-CA-V5 for 12 h. Table S12. mRNAs repressed in S2 cells expressing dGCN2-CA-V5 for 12 h. Table S13. GO term enrichment of mRNAs induced in S2 cells expressing dGCN2-CA-V5 for 6 h. Table S14. GO term enrichment of mRNAs induced in S2 cells expressing dGCN2-CA-V5 for 12 h. Table S15. GO term enrichment of mRNAs repressed in S2 cells expressing dGCN2-CA-V5 for 12 h. (XLSX 284 kb

Additional file 2: of The integrated stress response regulates BMP signalling through effects on translation

Figure S2. crc is Drosophila ATF4. (A) HA-crc-expressing S2 lysates and matched samples incubated with λ phosphatase (λ ppase) were subjected to SDS-PAGE and transferred to nitrocellulose. Immunoblotting was performed using an anti-HA antibody. (B) The 5’UTR of crc transcript E: small upstream open reading frames (uORFs) in orange; coding sequence in red. (C) Luminescence signal of luciferase control (blue bars) or 5’UTR-crcE-luciferase reporter (red bars) expressed in HEK293T cells presented as the ratio of firefly/Renilla luminescence fold change compared to vehicle-treated samples. Cells were treated with the indicated concentrations of ISRIB and/or tunicamycin for 16 h. Mean ± standard error of the mean (SEM). n = 3. P value calculated using ANOVA with Bonferroni post hoc testing. (D) Representative photomicrographs of adult eyes. Gmr (gmrGAL4 driver control), gmr > perk (gmrGAL4 > UAS-perk), gmr > crc RNAi (gmrGAL4 > UAS-crc RNAi) and gmr > perk;crc RNAi (gmrGAL4 > UAS-crc RNAi;UAS-perk). Scale bar = 200 μm. (E) Representative photomicrographs (5× objective) of adult wings of the indicated genotypes. en (enGAL4 driver control), en > dicer2;ppp1r15 RNAi (enGAL4 > UAS-dicer2;UAS-ppp1r15 RNAi), en > dicer2;crc RNAi (enGAL4 > UAS-dicer2;UAS-crc RNAi) and en > dicer2;crc RNAi;ppp1r15 RNAi (enGAL4 > UAS-dicer2;UAS-crc RNAi;UAS-ppp1r15 RNAi). Lower panels are enlargements of the crossvein territories. Scale bars = 250 μm. (F) Quantification of ACV phenotype in (E). (G) Representative photomicrographs of adult wings of the indicated genotypes. nab (nabGAL4 driver control), nab > ppp1r15 RNAi (nabGAL4 > UAS-ppp1r15 RNAi), nab > crc RNAi (nabGAL4 > UAS-crc RNAi) and nab > ppp1r15 RNAi;crc RNAi (nabGAL4 > UAS-crc RNAi;UAS-ppp1r15 RNAi). Lower panels are enlargements of the crossvein territories. Scale bars = 250 μm. (H) In situ hybridisation of w1118 wing imaginal disc with sense or antisense probes to residues 1405–1900 of crc transcript A. (I) Representative photomicrographs of adult wings of the indicated genotypes. nab (nabGAL4 driver control), nab > crc (nabGAL4 > UAS-crcA). Lower panels are enlargements of the crossvein territories. Scale bar = 250 μm. (PDF 3370 kb

Physiological effects and subjective tolerability of prone positioning in COVID-19 and healthy hypoxic challenge.

BACKGROUND: Prone positioning has a beneficial role in coronavirus disease 2019 (COVID-19) patients receiving ventilation but lacks evidence in awake non-ventilated patients, with most studies being retrospective, lacking control populations and information on subjective tolerability. METHODS: We conducted a prospective, single-centre study of prone positioning in awake non-ventilated patients with COVID-19 and non-COVID-19 pneumonia. The primary outcome was change in peripheral oxygenation in prone versus supine position. Secondary outcomes assessed effects on end-tidal CO2, respiratory rate, heart rate and subjective symptoms. We also recruited healthy volunteers to undergo proning during hypoxic challenge. RESULTS: 238 hospitalised patients with pneumonia were screened; 55 were eligible with 25 COVID-19 patients and three non-COVID-19 patients agreeing to undergo proning - the latter insufficient for further analysis. 10 healthy control volunteers underwent hypoxic challenge. Patients with COVID-19 had a median age of 64 years (interquartile range 53-75). Proning led to an increase in oxygen saturation measured by pulse oximetry (SpO2) compared to supine position (difference +1.62%; p=0.003) and occurred within 10 min of proning. There were no effects on end-tidal CO2, respiratory rate or heart rate. There was an increase in subjective discomfort (p=0.003), with no difference in breathlessness. Among healthy controls undergoing hypoxic challenge, proning did not lead to a change in SpO2 or subjective symptom scores. CONCLUSION: Identification of suitable patients with COVID-19 requiring oxygen supplementation from general ward environments for awake proning is challenging. Prone positioning leads to a small increase in SpO2 within 10 min of proning though is associated with increased discomfort