Jak2 V617F clonal hematopoiesis promotes arterial thrombosis via platelet activation and cross talk.

ABSTRACT: JAK2 V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre-mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.

Role of antimicrobial peptide cathelicidin in thrombosis and thromboinflammation.

Thrombosis is a frequent cause of cardiovascular mortality and hospitalization. Current antithrombotic strategies, however, target both thrombosis and physiological hemostasis and thereby increase bleeding risk. In recent years the pathophysiological understanding of thrombus formation has significantly advanced and inflammation has become a crucial element. Neutrophils as most frequent immune cells in the blood and their released mediators play a key role herein. Neutrophil-derived cathelicidin next to its strong antimicrobial properties has also shown to modulates thrombosis and thus presents a potential therapeutic target. In this article we review direct and indirect (immune- and endothelial cell-mediated) effects of cathelicidin on platelets and the coagulation system. Further we discuss its implications for large vessel thrombosis and consecutive thromboinflammation as well as immunothrombosis in sepsis and COVID-19 and give an outlook for potential therapeutic prospects.

Neutrophil "plucking" on megakaryocytes drives platelet production and boosts cardiovascular disease.

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.

Probing the chemical constituents of Cassia javanica and its in vitro analyses as a potent drug.

The phytochemical screening of the crude methanolic stem extracts of Cassia javanica plant showed the presence of different classes of organic compounds like alkaloids, tannins, flavonoids, saponins, phlobatanins, steroids, anthraquinone and cardiac glycoside. The starching frequencies of these functional groups were determined from FT-IR spectroscopic data. The crude and their fractions were examined for antibacterial potential against Klebsiella pneumoniae and Proteus mirabilis. The antibacterial assay showed maximum zone of inhibition for ethyl acetate fraction, i.e. 20 mm against Proteus mirabilis and 18 mm against Klebsiella pneumoniae in the comparison with Levofloxacin used as standard (40 mm). Meanwhile for methanolic crude extract, the inhibition zone was recorded 14 mm against Klebsiella pneumoniae and 22 mm against Proteus mirabilis. The minimum inhibitory concentrations and minimum bactericidal concentrations were recorded as 187.5 µg ml-1 against Proteus mirabilis and 93.75 µg ml-1 against Klebsiella pneumoniae. The scavenging free radical assay was noted as 69.61% at the concentration of 100 ppm.

COVID-19 highlights the model dilemma in biomedical research.

Scientists worldwide struggle to identify suitable animal models to study SARS-CoV-2 infections. Interspecies-related differences, such as host specificity, divergent immune responses, or the unavailability of species-specific reagents hamper the research. Human-based models, such as micro-engineered multi-organs-on-chip, may hold the solution.

Enhanced Systemic Anti-Angiogenic siVEGF Delivery Using PEGylated Oligo-d-arginine.

Angiogenesis mainly mediated by upregulation of vascular endothelial growth factor (VEGF) provides a hallmark of rapidly proliferating tumor cells and an essential component of the tumor growth and microenvironment, making it a targetable process for antitumor therapy. RNA interference (RNAi) provides a very effective tool for developing antitumor therapies; however, its application to date has been hampered due to the lack of efficient small interfering RNA (siRNA) delivery systems in vivo. Here, we report a polymeric gene carrier system based on PEGylation of a cationic cysteine-ended 9-mer arginine oligopeptide (CR9C), which provides effective siRNA systemic delivery and specifically suppresses VEGF (siVEGF). The PEG500-CR9C/siVEGF oligopeptoplex provided improved blood circulation, enhanced protection from serum proteases, reduced uptake in the liver and kidneys, enhanced tumor targeting, and down-regulated intratumoral VEGF level, which comprehensively resulted in improved antitumor efficacy without significant toxicity in vivo. PEG500-CR9C has a great potential for safe and efficient siRNA delivery with diverse applications.

Amelioration of atherosclerotic inflammation and plaques via endothelial adrenoceptor-targeted eNOS gene delivery using redox-sensitive polymer bearing l-arginine.

Endothelial dysfunction combined with inflammation leads to atherosclerosis. Endothelium-specific delivery of therapeutic agents at the cellular level-specifically in vivo-is still a difficult task for proper management of atherosclerosis. We designed a redox-sensitive poly(oligo-l-arginine) (rsPOLA) playing dual roles as an endothelium α-2 adrenoceptors(α-2ARs)-targeted gene carrier and as a substrate for endothelial nitric oxide synthase (eNOS). Overexpression of α-2ARs on atherosclerotic endothelial cells was confirmed and the eNOS/rsPOLA nanoplexes following systemic injection demonstrated to 1) enhance eNOS gene delivery into endothelial cells via α-2ARs/l-arginine specific binding, 2) increase intracellular level of nitric oxide, 3) suppress inflammatory response in endothelium and finally 4) reduce atherosclerotic plaque in a Ldlr-/- atherosclerotic mouse model. Among the tested nanoplexes [eNOS/rsPOLA, eNOS/{poly(oligo-d-arginine), rsPODA} and eNOS/(racemic mixture, rsRM)], eNOS/rsPOLA reduced atherosclerotic inflammation most effectively as we hypothesized. Current treatment strategy provides strong potential for further development of a gene therapeutic system to ameliorate inflammation and progressive atherosclerotic plaques.

RNAi-mediated silencing of TNF-α converting enzyme to down-regulate soluble TNF-α production for treatment of acute and chronic colitis.

Elevated level of tumor necrosis factor-α (TNF-α), one of the inflammatory cytokines, is considered to be a potential target for the inflammatory bowel disease (IBD) therapy. Recently, TNF-α converting enzyme (TACE), a sheddase playing an important role in cleaving and releasing bioactive soluble TNF-α, has been challenged with inhibitors to treat inflammatory diseases. Here, we report a novel anti-TNF-α strategy using a short hairpin RNA silencing TACE (shTACE) to prevent and treat colitis. The shTACE formed stable complexes with nona-arginine-based bio-cleavable disulfide bond-linked poly (arginine) (PAs-s) for enhanced gene delivery. Systemically administered shTACE/PAs-s peptoplexes efficiently decreased TNF-α levels, increased survival and alleviated pathophysiological parameters representing colitis severity. Our results demonstrate effectiveness and safety of shTACE/PAs-s peptoplexes with the capacity of overcoming acute and chronic ulcerative colitis through modulation of excessive inflammatory responses in the colon, providing a strong potential as a therapeutic agent for a broad variety of inflammatory diseases.

Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

Opinion of Pakistani physiotherapists/students about anatomy as a subject and method of teaching anatomy: a cross sectional survey.

OBJECTIVE: To recognise and identify the importance of Anatomy as a subject for physiotherapists/students. METHODS: The cross-sectional descriptive study was conducted from January to October 2011 at Riphah International University, Lahore, and comprised physiotherapists registered with Pakistan Physical Therapy Association and students pursuing undergraduate physiotherapy studies at the University. A survey proforma consisting of six questions was used for data collection which was analysed using SPSS 17. RESULTS: Of the 500 questionnaires distributed among physiotherapists and students, 286(57.2%) were collected back duly filled. Overall, 280(97.9%) respondents said Anatomy was important in physical therapy, 179(62.58%) said Anatomy was a difficult subject to retain knowledge of, 155 (54.38%) were of the view that only Clinical Anatomy should be studied, 204 (71.32%) were in favour of performing dissection, 259 (90.55%) found that teaching videos were essential for the study of Anatomy, and 89 (31.11%) believed Anatomy could be memorised by multiple readings. CONCLUSION: Despite being a difficult subject, Anatomy is an essential subject for medical professionals.

Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects.

Exploring N(1)-p-fluorobenzyl-cymserine as an inhibitor of 5-lipoxygenase as a candidate for type 2 diabetes and neurodegenerative disorder treatment.

Developing a single selective ligand to a target relevant to two mechanistically interlinked diseases, such as type 2 diabetes mellitus (T2DM) and a neurodegenerative disorder, like Parkinson's disease or Alzheimer's disease, provides the potential for an effective treatment that may impact both. The enzyme 5-lipoxygenase (5-LOX) has been revealed responsible for producing fatty acid molecules, leukotrienes. These leukotrienes are known to produce inflammatory responses in asthma and allergic reactions, to induce a reduction of tyrosine hydroxylase in brain, and are involved in the development of cardiac strokes, obesity and type 2 diabetes. N(1)-p-fluorobenzyl-cymserine (FBC), an analogue of cymserine and a known cholineterase inhibitor, was evaluated for inhibition of pleiotropic 5-LOX in our study. The stable 3D structure of 5-LOX was obtained from the Protein Data Bank (PDB) database and was implied for homology modeling of four reported mutant models. Each generated model was submitted to the Protein Model Database (PMDB) and employed for measuring inhibition and ligand efficiency of FBC with support of molecular docking. For each model, normal as well as mutant, FBC yielded remarkable inhibition constant values, with exothermic free binding energies. The current study revealed a highly reactive narrow fissure near the non-heme iron binding pocket of 5-LOX that contains residues crucial for 5-LOX stability and FBC binding. Investigating the binding of FBC with stabilized and destabilized 5-LOX structures confirmed it as a candidate therapeutic inhibitor worthy of assessment in preclinical models of T2DM and neurodegeneration.

Synthesis, spectroscopic and radical scavenging studies of palladium(II)-hydrazide complexes.

In present study, a series of palladium(II) complexes with biologically active hydrazide ligands have been synthesized, characterized and screened for their antioxidant (superoxide and DPPH radical scavenging) properties. Spectral studies (FT-IR, EI-mass, (13)C and (1)H NMR spectroscopy) and physico-chemical measurements including elemental analysis, magnetic susceptibility and conductivity measurements represented square planar structure for all complexes. Substituted and unsubstituted benzohydrazides (1-4) have shown monodentate behavior forming complexes of general formula [PdL2Cl2]. However, pyridinecarbohydrazides (5 and 6) were coordinated in bidentate fashion of [PdLCl2] general formula producing stable five-membered chelate ring. All palladium complexes were found to be considerably more potent inhibitors of DPPH free radical compared to free hydrazides. These complexes are even stronger DPPH scavengers than standard antioxidant propyl gallate. The complexes have also shown good superoxide scavenging ability compared to inactive free hydrazides, however complexes are weaker superoxide scavengers than ascorbic acid, a standard superoxide inhibitor. An interesting structure activity relationship has been evaluated.

Chemotherapeutic potential of orally administered poly(lactide-co-glycolide) microparticles containing isoniazid, rifampin, and pyrazinamide against experimental tuberculosis.

Poly(lactide-co-glycolide) (PLG) microspheres (diameter, less than 3 microm) containing isoniazid, rifampin, and pyrazinamide were used in a sustained oral drug delivery system to treat murine tuberculosis. Drug levels above the MIC were observed up to 72 h in plasma and for 9 days in various organs. Relative bioavailability of encapsulated drugs was greater than that of free drugs. Chemotherapy results showed better or equivalent clearance of bacilli in the PLG-drug-administered group (weekly) than with free drugs (daily).