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Parasite of the Month: Plasmodium vivax
No full textPlasmodium vivax is the most widely distributed of several plasmodial species that cause malaria, a disease associated with blood stage parasite replication. About 2.5 billion people are at risk of P. vivax infection, living mainly in Southeast Asia and the Americas, where P. vivax accounts for approximately 72% of malaria cases. In Africa, widespread lack of the Duffy antigen constrains transmission. The dormant liver form of the parasite, the hypnozoite, which can reactivate long after the primary infection and give rise to a relapsing blood stage infection, complicates eradication. In fact, hypnozoites are the origin of the majority of active blood stage infections. Primaquine and tafenoquine are the only drugs that prevent relapse; however, neither can be used during pregnancy or by people with glucose-6-phosphate dehydrogenase deficiency; and tafenoquine is not yet approved in children. Thus, this species of malaria-causing parasite is a unique challenge for eradication campaigns
S1P-S1PR1 activity controls VEGF-A signaling during lymphatic vessel development
No full textSphingosine-1-phosphate (S1P), a lipid signaling molecule produced by endothelial cells, is required for development and homeostasis of blood vessels. However, its role during lymphatic vessel development is unclear. We show in murine newborns that pharmaco-logically enhanced S1P signaling increases VEGF-A-dependent LEC proliferation. In contrast, S1PR1 inhibition, mediated by the antagonist NIBR0213 or LEC-specific genet-ic deletion of S1pr1, promotes filopodia formation and vessel branching, independent of VEGF-A. To investigate the S1P and VEGF-A signaling crosstalk observed in vivo, we used LECs cultured in vitro. We demonstrate that S1P activates endogenous S1PR1 in a constitutive, autocrine manner. Importantly, S1P-S1PR1 activity was required for VEGF-A-induced LEC proliferation and strongly supported ERK1/2 activation and VEGFR-2 trafficking to the perinuclear area. In conclusion, S1P-S1PR1 signaling promotes VEGF-A-dependent LEC proliferation and limits migratory and filopodia-forming responses. Hence, S1P-S1PR1 signaling is required for balanced growth factor-induced lymphangi-ogenesis and correctly patterned lymphatic vessels during postnatal development
Metabolic labeling of phospholipids in intact bacteria enables a fluorescence assay to detect loss of outer membrane asymmetry
No full textGram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of asymmetry due to mutation in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes PLs to be externalized to the outer leaflet of the OM and leads to OM permeability defects. Here, we employ metabolic labeling to detect externalized PLs on intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and the incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by liquid chromatography-mass spectrometry (LC-MS). AEPC readily reacted with a fluorescent copper-free click reagent in lysed cells, but poorly labeled intact wild-type cells. Fluorescence microscopy and flow cytometry analysis confirmed significantly higher levels of externalized AEPC were present on an E. coli LPS transport mutant (lptD4213) and a LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect PL externalization, dissect the mechanisms of PL retrograde and anterograde transport, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport
Discovery of the extracytoplasmic function σ factors
No full textThis special issue of Molecular Microbiology marks the 25th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one- and two-component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti-σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research
Fouling of Flow Reactors in Organolithium Mediated Transformations: Experience on Scale-up and Proposed Solution
No full textContinuous processing has been demonstrated to be a superior approach when applied to fast and energetic chemical transformations. Indeed, whereas classical batch or semi-batch methods require cryogenic conditions and slow addition rates of reactive chemistries, flow technologies enable a rapid mixing of synthetic partners in a highly controlled environment. As a result, low yielding and dangerous processes in batch can be performed at scale in a cost competitive and safer continuous modality. Despite high qualities and safety are common features, the perennial problem of solids build-up and pipes fouling always threatens the robustness and reliability of flow processes. Herein, a new methodology to prevent reactor clogging has been reported and discussed. The implementation of this new finding has been decisive to solve fouling issues encountered during the piloting of an organolithium based flow process
Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase
No full textThe development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC50 curve was generated
Un premier pas vers l’étude du collagène du cartilage articulaire en microscopie électronique
No full textLe cartilage articulaire est localisé à la surface des os impliqués dans une articulation et permet des mouvements avec un minimum de friction tout en agissant comme surface supportant les charges. Etant donné le nombre croissant de patients souffrant de pathologies telles que l’ostéoarthrite et l’arthrite rhumatoïde, l’étude du cartilage articulaire prend de plus en plus d’importance. Le cartilage articulaire est un sujet de choix pour être étudié par une technique d’imagerie de haute résolution comme la technique de microscopie électronique à transmission (MET), mais cette approche est complexe dû à la prépondérance de la matrice extracellulaire, composée essentiellement de protéoglycanes et de collagène et très difficile de préserver et colorer pour en extraire des informations sur l’ultrastructure. Afin de vérifier notre méthodologie pour reproduire des données déjà disponibles sur l’imagerie du cartilage par MET, et ainsi corréler les images avec le contenu en collagène, nous avons appliqué la MET à l’étude du ménisque et du plateau cartilagineux fémoral d’une articulation de la patte postérieure d’un rat adulte. Grâce à la préparation classique du tissu pour la MET focalisant sur la préservation du collagène au détriment des protéoglycanes, nous avons pu reproduire les données publiées et différencier les deux tissus en fonction de leur type de collagène prépondérant. La prochaine étape de notre travail sera d’utiliser ces images de MET pour valider les images de microscopie électronique à balayage (MEB) que nous aimerions générer à partir des mêmes tissus
Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo
No full textPeptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development
Tyrosine Kinase Inhibitor Gold Nanoconjugates for the Treatment of Non-Small Cell Lung Cancer.
No full textGold nanoparticles (AuNPs) have emerged as promising drug delivery candidates that can be leveraged for cancer therapy. Lung cancer (LC) is a heterogeneous disease that imposes a significant burden on society, with an unmet need for new therapies. Chemotherapeutic drugs such as afatinib (Afb), which is clinically approved for the treatment of epidermal growth factor receptor positive LC, is hydrophobic and has low bioavailability leading to spread around the body, causing severe side effects. Herein, we present a novel afatinib-AuNP formulation termed Afb-AuNPs, with the aim of improving drug efficacy and biocompatibility. This was achieved by synthesis of an alkyne-bearing Afb derivative and reaction with azide-functionalized lipoic acid using copper-catalyzed click chemistry, then conjugation to AuNPs via alkylthiol-gold bond formation. The Afb-AuNPs were found to possess up to 3.7-fold increased potency when administered to LC cells in vitro and were capable of significantly inhibiting cancer cell proliferation, as assessed by MTT assay and electric cell-substrate impedance sensing, respectively. Furthermore, when exposed to Afb-AuNPs, human alveolar epithelial type I-like cells, a model of the healthy lung epithelium, maintained viability and were found to release less proinflammatory cytokines when compared to free drug, demonstrating the biocompatibility of our formulation. This study provides a new platform for the development of nontraditional AuNP conjugates which can be applied to other molecules of therapeutic or diagnostic utility, with potential to be combined with photothermal therapy in other cancers
Nanotechnology in the diagnosis and treatment of lung cancer.
No full textLung cancer is an umbrella term for a subset of heterogeneous diseases that are collectively responsible for the most cancer-related deaths worldwide. Despite the tremendous progress made in understanding lung tumour biology, advances in early diagnosis, multimodal therapy and deciphering molecular mechanisms of drug resistance, overall curative outcomes remain low, especially in metastatic disease. Nanotechnology, in particular nanoparticles (NPs), continue to progressively impact the way by which tumours are diagnosed and treated. The unique physicochemical properties of materials at the nanoscale grant access to a diverse molecular toolkit that can be manipulated for use in respiratory oncology. This realisation has resulted in several clinically approved NP formulations and many more in clinical trials. However, NPs are not a panacea and have yet to be utilised to maximal effect in lung cancer, and medicine in a wider context. This review serves to: describe the complexity of lung cancer, the current diagnostic and therapeutic environment, and highlight the recent advancements of nanotechnology based approaches in diagnosis and treatment of respiratory malignancies. Finally, a brief outlook on the future directions of nanomedicine is provided; presently the full potential of the field is yet to be realised. By gleaning lessons and integrating advancements from neighbouring disciplines, nanomedicine can be elevated to a position where the current barriers that stymie full clinical impact are lifted