Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide.

During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s-1) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s-1). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s-1) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.

A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution.

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P2Y1, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes.

Thrombosis and Hemodynamics: external and intrathrombus gradients.

Distinct from dilute, isotropic, and homogeneous reaction systems typically used in laboratory kinetic assays, blood is concentrated, two-phase, flowing, and highly anisotropic when clotting on a surface. This review focuses on spatial gradients that are generated and can dictate thrombus structure and function. Novel experimental and computational tools have recently emerged to explore reaction-transport coupling during clotting. Multiscale simulations help bridge tissue length scales (the coronary arteries) to millimeter scales of a growing clot to the microscopic scale of single-cell signaling and adhesion. Microfluidic devices help create and control pathological velocity profiles, albeit at a low Reynolds number. Since rate processes and force loading are often coupled, this review highlights prevailing convective-diffusive transport physics that modulate cellular and molecular processes during thrombus formation.

Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models.

Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.

Neutralization of PD-L2 is Essential for Overcoming Immune Checkpoint Blockade Resistance in Ovarian Cancer.

PURPOSE: Ovarian cancer represents a major clinical hurdle for immune checkpoint blockade (ICB), with reported low patient response rates. We found that the immune checkpoint ligand PD-L2 is robustly expressed in patient samples of ovarian cancers and other malignancies exhibiting suboptimal response to ICB but not in cancers that are ICB sensitive. Therefore, we hypothesize that PD-L2 can facilitate immune escape from ICB through incomplete blockade of the PD-1 signaling pathway. EXPERIMENTAL DESIGN: We engineered a soluble form of the PD-1 receptor (sPD-1) capable of binding and neutralizing both PD-L2 and PD-L1 with ×200 and ×10,000 folds improvement in binding affinity over wild-type PD-1 leading to superior inhibition of ligand-mediated PD-1 activities. RESULTS: Both in vitro and in vivo analyses performed in this study demonstrated that the high-affinity sPD-1 molecule is superior at blocking both PD-L1- and PD-L2-mediated immune evasion and reducing tumor growth in immune-competent murine models of ovarian cancer. CONCLUSIONS: The data presented in this study provide justification for using a dual targeting, high-affinity sPD-1 receptor as an alternative to PD-1 or PD-L1 therapeutic antibodies for achieving superior therapeutic efficacy in cancers expressing both PD-L2 and PD-L1.

Routine surveillance of pelvic and lower extremity deep vein thrombosis in stroke patients with patent foramen ovale.

Patent foramen ovale (PFO) is a potential conduit for paradoxical embolization to the systemic atrial circulation of a thrombus originating in the venous system. In a selected group of subjects, the prevalence of deep vein thrombosis (DVT) was assessed. Subjects were identified if they underwent magnetic resonance venography (MRV) pelvis and lower extremity doppler (LE-VDU) for assessment of DVT with PFO. The primary outcome measure was to report the number of patients with paradoxical embolization as their suspected etiology of stroke due to the presence of DVT, which then will be considered as determined stroke. Others with determined stroke diagnosis were reported using Treatment of Acute Stroke Trial (TOAST) criteria. At discharge, those without etiology of their stroke were grouped under embolic stroke of undetermined source (ESUS). We further analyzed the prevalence of DVT by age group, ≤ 60 years vs > 60 years to describe if the prevalence is higher with younger age and to evaluate if higher Risk of Paradoxical Embolism (ROPE) score will have higher number of DVTs compared to lower ROPE scores. Of the 293, 19 (7%) were strokes due to paradoxical embolism. At discharge, determined stroke were 54% vs ESUS were 46%. The overall prevalence of DVT was 19 (7%); MRV-pelvis 13 (4%), and LE-VDU was 9 (3%). No significant difference was noted using both modalities. However, in multivariable regression analysis, a trend suggested an association between pelvic thrombi and high ROPE score as the etiology of stroke; OR 3.56 (0.98, 12.93); p = 0.054. Detection of DVT was not associated with PFO, high ROPE scores or young age. Our data indicate an over-reliance of testing for DVT, particularly MRV pelvis with contrast, in patients with PFO. Clinical studies are needed to identify other factors predictive of DVT in patients with ischemic stroke and PFO.

The m6A RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor.

Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

Generation of Stable Expression Mammalian Cell Lines Using Lentivirus.

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. The advantage of using the 3rd generation lentivirus are that are very safe and they are replication incompetent.

Endovascular Thrombectomy for Mild Strokes: How Low Should We Go?

Background and Purpose- Endovascular thrombectomy (EVT) is effective for acute ischemic stroke with large vessel occlusion and National Institutes of Health Stroke Scale (NIHSS) ≥6. However, EVT benefit for mild deficits large vessel occlusions (NIHSS, <6) is uncertain. We evaluated EVT efficacy and safety in mild strokes with large vessel occlusion. Methods- A retrospective cohort of patients with anterior circulation large vessel occlusion and NIHSS <6 presenting within 24 hours from last seen normal were pooled. Patients were divided into 2 groups: EVT or medical management. Ninety-day mRS of 0 to 1 was the primary outcome, mRS of 0 to 2 was the secondary. Symptomatic intracerebral hemorrhage was the safety outcome. Clinical outcomes were compared through a multivariable logistic regression after adjusting for age, presentation NIHSS, time last seen normal to presentation, center, IV alteplase, Alberta Stroke Program early computed tomographic score, and thrombus location. We then performed propensity score matching as a sensitivity analysis. Results were also stratified by thrombus location. Results- Two hundred fourteen patients (EVT, 124; medical management, 90) were included from 8 US and Spain centers between January 2012 and March 2017. The groups were similar in age, Alberta Stroke Program early computed tomographic score, IV alteplase rate and time last seen normal to presentation. There was no difference in mRS of 0 to 1 between EVT and medical management (55.7% versus 54.4%, respectively; adjusted odds ratio, 1.3; 95% CI, 0.64-2.64; P=0.47). Similar results were seen for mRS of 0 to 2 (63.3% EVT versus 67.8% medical management; adjusted odds ratio, 0.9; 95% CI, 0.43-1.88; P=0.77). In a propensity matching analysis, there was no treatment effect in 62 matched pairs (53.5% EVT, 48.4% medical management; odds ratio, 1.17; 95% CI, 0.54-2.52; P=0.69). There was no statistically significant difference when stratified by any thrombus location; M1 approached significance ( P=0.07). Symptomatic intracerebral hemorrhage rates were higher with thrombectomy (5.8% EVT versus 0% medical management; P=0.02). Conclusions- Our retrospective multicenter cohort study showed no improvement in excellent and independent functional outcomes in mild strokes (NIHSS, <6) receiving thrombectomy irrespective of thrombus location, with increased symptomatic intracerebral hemorrhage rates, consistent with the guidelines recommending the treatment for NIHSS ≥6. There was a signal toward benefit with EVT only in M1 occlusions; however, this needs to be further evaluated in future randomized control trials.

Cross-talk between chromatin acetylation and SUMOylation of tripartite motif-containing protein 24 (TRIM24) impacts cell adhesion.

Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers. Numerous interactions between specific reader protein domains and histone PTMs and their regulatory outcomes have been reported, but little is known about how reader proteins may in turn be modulated by these interactions. Tripartite motif-containing protein 24 (TRIM24) is a histone reader aberrantly expressed in multiple cancers. Here, our investigation revealed functional cross-talk between histone acetylation and TRIM24 SUMOylation. Binding of TRIM24 to chromatin via its tandem PHD-bromodomain, which recognizes unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/K23ac), led to TRIM24 SUMOylation at lysine residues 723 and 741. Inactivation of the bromodomain, either by mutation or with a small-molecule inhibitor, IACS-9571, abolished TRIM24 SUMOylation. Conversely, inhibition of histone deacetylation markedly increased TRIM24's interaction with chromatin and its SUMOylation. Of note, gene expression profiling of MCF7 cells expressing WT versus SUMO-deficient TRIM24 identified cell adhesion as the major pathway regulated by the cross-talk between chromatin acetylation and TRIM24 SUMOylation. In conclusion, our findings establish a new link between histone H3 acetylation and SUMOylation of the reader protein TRIM24, a functional connection that may bear on TRIM24's oncogenic function and may inform future studies of PTM cross-talk between histones and epigenetic regulators.

Exciton Emission Intensity Modulation of Monolayer MoS2 via Au Plasmon Coupling.

Modulation of photoluminescence of atomically thin transition metal dichalcogenide two-dimensional materials is critical for their integration in optoelectronic and photonic device applications. By coupling with different plasmonic array geometries, we have shown that the photoluminescence intensity can be enhanced and quenched in comparison with pristine monolayer MoS2. The enhanced exciton emission intensity can be further tuned by varying the angle of polarized incident excitation. Through controlled variation of the structural parameters of the plasmonic array in our experiment, we demonstrate modulation of the photoluminescence intensity from nearly fourfold quenching to approximately threefold enhancement. Our data indicates that the plasmonic resonance couples to optical fields at both, excitation and emission bands, and increases the spontaneous emission rate in a double spacing plasmonic array structure as compared with an equal spacing array structure. Furthermore our experimental results are supported by numerical as well as full electromagnetic wave simulations. This study can facilitate the incorporation of plasmon-enhanced transition metal dichalcogenide structures in photodetector, sensor and light emitter applications.

Regulation of gene expression in human cancers by TRIM24.

Tripartite Motif-containing protein 24 (TRIM24) functions as an E3 ligase targeting p53 for ubiquitination, a histone 'reader' that interacts with a specific signature of histone post-translational modifications and a co-regulator of nuclear receptor-regulated transcription. Although mouse models of Trim24 depletion suggest that TRIM24 may be a liver-specific tumor suppressor, several studies show that human TRIM24 is an oncogene when aberrantly over expressed. This review focuses on the mechanisms of TRIM24 functions in oncogenesis and metabolic reprogramming, which underlie recent interest in therapeutic targeting of aberrant TRIM24 in human cancers.

Acute splenic responses in patients with ischemic stroke and intracerebral hemorrhage.

Animal models provide evidence of spleen mediated post-stroke activation of the peripheral immune system. Translation of these findings to stroke patients requires estimation of pre-stroke spleen volume along with quantification of its day-to-day variation. We enrolled a cohort of 158 healthy volunteers and measured their spleen volume over the course of five consecutive days. We also enrolled a concurrent cohort of 158 stroke patients, measured initial spleen volume within 24 h of stroke symptom onset followed by daily assessments. Blood samples for cytokine analysis were collected from a subset of patients. Using data from healthy volunteers, we fit longitudinal quantile regression models to construct gender and body surface area based normograms of spleen volume. We quantified day-to-day variation and defined splenic contraction. Based on our criteria, approximately 40% of stroke patients experienced substantial post-stroke reduction in splenic volume. African Americans, older patients, and patients with past history of stroke have significantly higher odds of post-stroke splenic contraction. All measured cytokine levels were elevated in patients with splenic contraction, with significant differences for interferon gamma, interleukin 6, 10, 12, and 13. Our work provides reference standards for further work, validation of pre-clinical findings, and characterization of patients with post-stroke splenic contraction.

Non invasive prenatal diagnosis of aneuploidy: next generation sequencing or fetal DNA enrichment?

Current invasive procedures [amniocentesis and chorionic villus sampling (CVS)] pose a risk to mother and fetus and such diagnostic procedures are available only to high risk pregnancies limiting aneuploidy detection rate. This review seeks to highlight the necessity of investing in non invasive prenatal diagnosis (NIPD) and how NIPD would improve patient safety and detection rate as well as allowing detection earlier in pregnancy. Non invasive prenatal diagnosis can take either a proteomics approach or nucleic acid-based approach; this review focuses on the latter. Since the discovery of cell free fetal DNA (cffDNA) and fetal RNA in maternal plasma, procedures have been developed for detection for monogenic traits and for some have become well established (e.g., RHD blood group status). However, NIPD of aneuploidies remains technically challenging. This review examines currently published literature evaluating techniques and approaches that have been suggested and developed for aneuploidy detection, highlighting their advantages and limitations and areas for further research.

Endoscopic ultrasound-guided fine-needle aspiration cytology in the evaluation of suspected tuberculosis in patients with isolated mediastinal lymphadenopathy.

BACKGROUND AND STUDY AIMS: Patients with suspected tuberculosis without pulmonary lesions and with mediastinal lymphadenopathy often pose a diagnostic challenge. Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) cytology is an established modality to evaluate mediastinal and abdominal lesions. The aim of the present study was to evaluate the role of EUS-FNA in isolated mediastinal lymphadenopathy in patients suspected of having tuberculosis. METHODS: Consecutive patients suspected of having tuberculosis with isolated mediastinal lymphadenopathy were included in a prospective study. Mediastinal lymphadenopathy was diagnosed on a contrast-enhanced computed tomography scan of the chest. Patients with concomitant lung parenchymal lesions were excluded. Previous attempts to diagnose the etiology of lymphadenopathy had failed in 69 % of patients. EUS-FNA was performed on an outpatient basis under conscious sedation. The sensitivity, specificity, and diagnostic accuracy of EUS-FNA were calculated. RESULTS: A total of 60 consecutive patients (mean age 39.8 years, 58 % males) with mediastinal lymphadenopathy were included. EUS confirmed the presence of mediastinal lymph nodes ranging in size from 8 mm to 40 mm (mean 26 mm) in all patients. EUS-FNA provided an adequate tissue sample in 54 patients during the first examination and repeat EUS-FNA was necessary in six patients. A final diagnosis was obtained by EUS-FNA in 42 patients (tuberculosis in 32, sarcoidosis in six, and Hodgkin's disease in four patients). An additional 14 patients were treated for tuberculosis based on EUS-FNA and clinical features. Mediastinoscopy was required for diagnosis in the remaining four patients. EUS-FNA had an overall diagnostic yield of 93 %, sensitivity of 71 %, specificity of 100 %, and positive predictive value of 100 %. CONCLUSION: EUS-FNA is an accurate, safe, and minimally invasive modality for evaluating isolated mediastinal lymphadenopathy in patients suspected of having tuberculosis in an endemic area with a high prevalence of tuberculosis.

Biological synthesis of metallic nanoparticles.

The synthesis of metallic nanoparticles is an active area of academic and, more importantly, "application research" in nanotechnology. A variety of chemical and physical procedures could be used for synthesis of metallic nanoparticles. However, these methods are fraught with many problems including use of toxic solvents, generation of hazardous by-products, and high energy consumption. Accordingly, there is an essential need to develop environmentally benign procedures for synthesis of metallic nanoparticles. A promising approach to achieve this objective is to exploit the array of biological resources in nature. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles. In this review, we provide an overview of various reports of synthesis of metallic nanoparticles by biological means. FROM THE CLINICAL EDITOR: This review provides an overview of various methods of synthesis of metallic nanoparticles by biological means. Many chemical and physical procedures used for synthesis of metallic nanoparticles are fraught with major problems: toxic solvents, hazardous by-products, high energy consumption. Over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles.

Synthesis, spectral, thermal and anti-fungal studies of organotin(IV) thiohydrazone complexes.

The reaction of tribenzyltin(IV) chloride and di(para-chlorobenzyl)tin(IV) dichloride with thiohydrazones derived by condensation of 2-phenylethyl N-thiohydrazide with benzaldehyde, salicaldehyde, p-methylacetophenone and cinnamaldehyde have been investigated in 1:1 molar ratio. These ligands act as neutral, bidentate species and coordinate to the central tin (IV) atom through the thiosulphur and azomethine nitrogen. The newly synthesized complexes have been characterized by elemental analysis and molecular weight determination. The mode of bonding of the complexes has been suggested on the basis of infrared, electronic and (1)H NMR spectroscopy, and probable structures have been assigned to these complexes. Phenomenological and kinetic parameters have been calculated using thermogravimetric (TG) and differential thermal analytical (DTA) curves and their variations have been correlated with some structural parameters of the complexes. The ligands and their tin(IV) complexes have been screened in vitro for their fungicidal activity against Rhizoctonia solanii and Sclerotium rolfsii and found to be quite active in this respect.