Silver Nanoparticles (AgNPs) Uptake by Caveolae-Dependent Endocytosis is Responsible for Their Selective Effect Towards Castration Resistant Prostate Cancer.

Purpose: Castration Resistant Prostate Cancer (CRPC) is characterized by poor prognosis and limited therapeutic options. AgNPs functionalized with glucose (G-AgNPs) were observed cytotoxic to CRPC cell lines (PC-3 and Du-145) and not LNCaP. This study aims to evaluate AgNPs and G-AgNPs' uptake mechanisms in these cells and understand their role in the selective effect against CRPC cells. Methods: Uptake of AgNPs and G-AgNPs was assessed through transmission electron microscopy (TEM). A microRNA (miRNAs) analysis approach was used to uncover the main molecular differences responsible for the endocytic mechanisms' regulation. Caveolin (Cav) 1 and 2 mRNA and protein levels were assessed in the three cell lines. Caveolae-dependent endocytosis was inhibited with genistein or siCav1- and siCav2- in PC-3 and Du-145 and resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration. Caveolae-dependent endocytosis was induced with Cav1+ and Cav2+ plasmids in LNCaP, resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration and TEM to assess their location. Results: AgNPs and G-AgNPs were not uptaked by LNCaP. miRNA analysis revealed 37 upregulated and 90 downregulated miRNAs. Functional enrichment analysis of miRNAs' targets resulted in enrichment of terms related to endocytosis and caveolae. We observed that Cav1 and Cav2 are not expressed in LNCaP. Inhibiting caveolae-dependent endocytosis in Du-145 and PC-3 led to a significative reduction of cytotoxic capacity of AgNPs and G-AgNPs and induction of caveolae-dependent endocytosis in LNCaP lead to a significative increase as well as their uptake by cells. Conclusion: This study shows the potential of these AgNPs as a new therapeutic approach directed to CRPC patients, uncovers caveolae-dependent endocytosis as the uptake mechanism of these AgNPs and highlights deregulation of Cav1 and Cav2 expression as a key difference in hormone sensitive and resistant PCa cells which may be responsible for drug resistance.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Bemarituzumab as first-line treatment for locally advanced or metastatic gastric/gastroesophageal junction adenocarcinoma: final analysis of the randomized phase 2 FIGHT trial.

BACKGROUND: We report the final results of the randomized phase 2 FIGHT trial that evaluated bemarituzumab, a humanized monoclonal antibody selective for fibroblast growth factor receptor 2b (FGFR2b), plus mFOLFOX6 in patients with FGFR2b-positive (2 + /3 + membranous staining by immunohistochemistry), HER-2-negative gastric or gastroesophageal junction cancer (GC). METHODS: Patients received bemarituzumab (15 mg/kg) or placebo once every 2 weeks with an additional bemarituzumab (7.5 mg/kg) or placebo dose on cycle 1 day 8. All patients received mFOLFOX6. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate, and safety. Efficacy was evaluated after a minimum follow-up of 24 months. RESULTS: In the bemarituzumab-mFOLFOX6 (N = 77) and placebo-mFOLFOX6 (N = 78) arms, respectively, 59.7% and 66.7% of patients were FGFR2b-positive in ≥ 10% of tumor cells. The median PFS (95% confidence interval [CI]) was 9.5 months (7.3-13.7) with bemarituzumab-mFOLFOX6 and 7.4 months (5.7-8.4) with placebo-mFOLFOX6 (hazard ratio [HR], 0.72; 95% CI 0.49-1.08); median OS (95% CI) was 19.2 (13.6-24.2) and 13.5 (9.3-15.9) months, respectively (HR 0.77; 95% CI 0.52-1.14). Observed efficacy in FGFR2b-positive GC in ≥ 10% of tumor cells was: PFS: HR 0.43 (95% CI 0.26-0.73); OS: HR 0.52 (95% CI 0.31-0.85). No new safety findings were reported. CONCLUSIONS: In FGFR2b-positive advanced GC, the combination of bemarituzumab-mFOLFOX6 led to numerically longer median PFS and OS compared with mFOLFOX6 alone. Efficacy was more pronounced with FGFR2b overexpression in ≥ 10% of tumor cells. Confirmatory phase 3 trials are ongoing (NCT05052801, NCT05111626). CLINICAL TRIAL REGISTRATION: NCT03694522.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review.

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

Bemarituzumab in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma (FIGHT): a randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Outcomes are poor in patients with HER2-negative, advanced gastric or gastro-oesophageal junction adenocarcinomas. In this study, we investigated efficacy and safety of the first-in-class, afucosylated, humanised IgG1 anti-fibroblast growth factor receptor 2 isoform IIb (FGFR2b) monoclonal antibody bemarituzumab with modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma. METHODS: In the randomised, double-blind, placebo-controlled phase 2 trial (FIGHT), patients aged 18 years and older with HER2 non-positive, FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma, and an Eastern Cooperative Oncology Group performance status of 0-1 were recruited from 144 clinical sites across 17 countries. Patients with previous treatment with any selective inhibitor of the FGF-FGFR pathway were excluded. Eligible patients were randomly assigned (1:1), using permuted-block randomisation (block size of four) and a central interactive voice-web-based response system, stratified by geographical region, previous treatment with curative intent, and administration of mFOLFOX6 while being screened for FGFR2b status, to either bemarituzumab (15 mg/kg of bodyweight) or matched placebo intravenously every 2 weeks. All patients also received mFOLFOX6 (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, and 5-fluorouracil as a 400 mg/m2 bolus followed by 2400 mg/m2 over approximately 46 h) intravenously every 2 weeks. Patients were given treatment until disease progression (defined by Response Evaluation Criteria in Solid Tumours [RECIST] version 1.1), unacceptable toxicity, withdrawal of consent, or death. The primary endpoint was progression-free survival in the intention-to-treat population (defined as all patients randomly assigned to treatment). Safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, NCT03694522, and is now complete. FINDINGS: Between Nov 14, 2017, and May 8, 2020, 910 patients were screened and 155 were randomly assigned to the bemarituzumab (n=77) or placebo group (n=78). Median age was 60·0 years (IQR 51·0-67·0), 44 (28%) participants were women, 111 (72%) were men, 89 (57%) were Asian, and 61 (39%) were White. At the time of the primary analysis and at a median follow-up of 10·9 months (IQR 6·3-14·2), median progression-free survival was 9·5 months (95% CI 7·3-12·9) in the bemarituzumab group and 7·4 months (5·8-8·4) in the placebo group (hazard ratio [HR] 0·68 [95% CI 0·44-1·04; p=0·073). Common grade 3 or worse adverse events were decreased neutrophil count (23 [30%] of 76 in the bemarituzumab group vs 27 [35%] of 77 in the placebo group), cornea disorder (18 [24%] vs none), neutropenia (ten [13%] vs seven [9%]), stomatitis (seven [9%] vs one [1%]), and anaemia (six [8%] vs ten [13%]). Serious treatment-emergent adverse events were reported in 24 (32%) patients in the bemarituzumab group and 28 (36%) in the placebo group. Serious mFOLFOX6 treatment-related adverse events occurred in nine (12%) patients in the bemarituzumab group and in 15 (19%) patients in the placebo group. All-grade corneal events (adverse events of special interest) occurred in 51 (67%) patients in the bemarituzumab group and eight (10%) in the placebo group; grade 3 corneal events were reported only in 18 (24%) patients in the bemarituzumab group. Treatment-related deaths occurred in three patients in the bemarituzumab group (two due to sepsis, one due to pneumonia) and none in the placebo group. INTERPRETATION: In this exploratory phase 2 study, despite no statistically significant improvement in progression-free survival, treatment with bemarituzumab showed promising clinical efficacy. Confirmatory phase 3 trials of bemarituzumab plus mFOLFOX6 powered to demonstrate statistical significance are being investigated in patients with previously untreated, FGFR2b-overexpressing, advanced gastric or gastro-oesophageal junction adenocarcinoma. FUNDING: Five Prime Therapeutics.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients.

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

Susceptibility of Sea Bream (Sparus aurata) to AIP56, an AB-Type Toxin Secreted by Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts.

Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification.

Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

Eucalyptus globulus Labill. decoction extract inhibits the growth of NCI-H460 cells by increasing the p53 levels and altering the cell cycle profile.

Eucalyptus globulus Labill. is a widespread evergreen plant belonging to the Myrtaceae family. Several species of Eucalyptus are known to have a plethora of medicinal properties, particularly anti-tumor activity, which prompts the study of the chemical composition and bioactivity of extracts from this plant. Hereby, the main aims of this work were to (i) profile the phenolic compounds in E. globulus extracts prepared by decoction and infusion; (ii) test the cell growth inhibitory activity of E. globulus decoction and infusion, in three human tumor cell line models: colorectal, pancreatic and non-small cell lung cancer (HCT-15, PANC-1 and NCI-H460, respectively); and (iii) study the mechanism of action of the most potent extract in the most sensitive cell line. Our work demonstrated that both the decoction and infusion preparations revealed the presence of phenolic acids, flavonoids and gallotannins, the last group being the most abundant polyphenols found, especially two digalloyl-glucosides. Both extracts inhibited the growth of all the tumor cell lines tested. The decoction extract was the most potent in inhibiting the NCI-H460 cell growth (lower GI50 determined by sulforhodamine B assay), which could be due to its higher content of phenolic compounds. Hence, the effect of the decoction extract on the NCI-H460 cells was further investigated. For this, cell viability (by Trypan blue exclusion assay), the cell cycle profile and apoptosis (by flow cytometry), cell proliferation (by bromodeoxyuridine assay) and protein expression (by western blot) were analyzed. Two different concentrations of the extract (52 µg mL-1 and 104 µg mL-1, corresponding to GI50 and 2 × GI50 concentration) were tested in these studies. Remarkably, the E. globulus decoction extract caused a dose-dependent decrease in the NCI-H460 cell number, which was correlated with a cell cycle arrest in the G0/G1 phase, a decrease in cell proliferation and an increase in the expression of p53, p21 and cyclin D1 proteins. Interestingly, no differences were found in the levels of ds-DNA damage and in the levels of apoptosis. This work highlights the relevance of the Eucalyptus globulus Labill. extract as a source of bioactive compounds with potential anti-tumor activity.

Epithelial (E)-Cadherin is a Novel Mediator of Platelet Aggregation and Clot Stability.

Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3ß (GSK3ß) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3ß signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.

Achillea millefolium L. hydroethanolic extract inhibits growth of human tumor cell lines by interfering with cell cycle and inducing apoptosis.

The cell growth inhibitory activity of the hydroethanolic extract of Achillea millefolium was studied in human tumor cell lines (NCI-H460 and HCT-15) and its mechanism of action was investigated. The GI50 concentration was determined with the sulforhodamine B assay and cell cycle and apoptosis were analyzed by flow cytometry following incubation with PI or Annexin V FITC/PI, respectively. The expression levels of proteins involved in cell cycle and apoptosis were analyzed by Western blot. The extracts were characterized regarding their phenolic composition by LC-DAD-ESI/MS. 3,5-O-Dicaffeoylquinic acid, followed by 5-O-caffeoylquinic acid, were the main phenolic acids, while, luteolin-O-acetylhexoside and apigenin-O-acetylhexoside were the main flavonoids. This extract decreased the growth of the tested cell lines, being more potent in HCT-15 and then in NCI-H460 cells. Two different concentrations of the extract (75 and 100 µg/mL) caused alterations in cell cycle profile and increased apoptosis levels in HCT-15 and NCI-H460 cells. Moreover, the extract caused an increase in p53 and p21 expression in NCI-H460 cells (which have wt p53), and reduced XIAP levels in HCT-15 cells (with mutant p53). This work enhances the importance of A. millefolium as source of bioactive phenolic compounds, particularly of XIAP inhibitors.

SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bß3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.

Gla-rich protein acts as a calcification inhibitor in the human cardiovascular system.

OBJECTIVE: Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K-dependent Gla-rich protein (GRP) in vascular and valvular calcification processes. APPROACH AND RESULTS: Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both γ-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP-fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion. CONCLUSIONS: GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A-MGP calcification inhibitory system. GRP activity was found to be dependent on its γ-carboxylation status, with potential clinical relevance.

Gla-rich protein is a potential new vitamin K target in cancer: evidences for a direct GRP-mineral interaction.

Gla-rich protein (GRP) was described in sturgeon as a new vitamin-K-dependent protein (VKDP) with a high density of Gla residues and associated with ectopic calcifications in humans. Although VKDPs function has been related with γ-carboxylation, the Gla status of GRP in humans is still unknown. Here, we investigated the expression of recently identified GRP spliced transcripts, the γ-carboxylation status, and its association with ectopic calcifications, in skin basal cell and breast carcinomas. GRP-F1 was identified as the predominant splice variant expressed in healthy and cancer tissues. Patterns of γ-carboxylated GRP (cGRP)/undercarboxylated GRP (ucGRP) accumulation in healthy and cancer tissues were determined by immunohistochemistry, using newly developed conformation-specific antibodies. Both GRP protein forms were found colocalized in healthy tissues, while ucGRP was the predominant form associated with tumor cells. Both cGRP and ucGRP found at sites of microcalcifications were shown to have in vitro calcium mineral-binding capacity. The decreased levels of cGRP and predominance of ucGRP in tumor cells suggest that GRP may represent a new target for the anticancer potential of vitamin K. Also, the direct interaction of cGRP and ucGRP with BCP crystals provides a possible mechanism explaining GRP association with pathological mineralization.

Dynamic migration and cell-cell interactions of early reprogramming revealed by high-resolution time-lapse imaging.

Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multicolor time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as 5 minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells that emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies, and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct two-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate that E-cadherin is required for proper cellular interactions from an early stage of reprogramming, including the two-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.

Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization.We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.