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To achieve high-sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.
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Citometria de Fluxo , Humanos , Citometria de Fluxo/métodos , Células K562 , Imunofenotipagem/métodos , Neoplasia Residual , Eritrócitos/citologia , Leucócitos/citologia , Contagem de Células/métodosRESUMO
The ability of an octanuclear cubic coordination cage to catalyse a nucleophilic aromatic substitution reaction on a cavity-bound guest was studied with 2,4-dinitrofluorobenzene (DNFB) as the guest/substrate. It was found that DNFB undergoes a catalysed reaction with hydroxide ions within the cavity of the cubic cage (in aqueous buffer solution, pHâ 8.6). The rate enhancement of kcat/kuncat was determined to be 22, with cavity binding of the guest being required for catalysis to occur. The product, 2,4-dinitrophenolate (DNP), remained bound within the cavity due to electrostatic stabilisation and exerts two apparently contradictory effects: it initially auto-catalyses the reaction when present at low concentrations, but at higher concentrations inhibits catalysis when a pair of DNP guests block the cavity. When encapsulated, the UV/Vis absorption spectrum of DNP is red-shifted when compared to the spectrum of free DNP in aqueous solution. Further investigations using other aromatic guests determined that a similar red-shift on cavity binding also occurred for 4-nitrophenolate (4NP) at pHâ 8.6. The red-shift was used to determine the stoichiometry of guest binding of DNP and 4NP within the cage cavity, which was confirmed by structural analysis with X-ray crystallography; and was also used to perform catalytic kinetic studies in the solution-state.
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Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.
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Adenina/análogos & derivados , Curcumina , Leucemia Linfocítica Crônica de Células B , Piperidinas , Vidarabina/análogos & derivados , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Curcumina/farmacologia , Curcumina/uso terapêutico , TetraploidiaRESUMO
Aberrant crystallization within the human body can lead to several disease states or adverse outcomes, yet much remains to be understood about the critical stages leading to these events, which can include crystal nucleation and growth, crystal aggregation, and the adhesion of crystals to cells. Kidney stones, which are aggregates of single crystals with physiological origins, are particularly illustrative of pathological crystallization, with 10% of the U.S. population experiencing at least one stone occurrence in their lifetimes. The human record of kidney stones is more than 2000 years old, as noted by Hippocrates in his renowned oath and much later by Robert Hooke in his treatise Micrographia. William Hyde Wollaston, who was a physician, chemist, physicist, and crystallographer, was fascinated with stones, leading him to discover an unusual stone that he described in 1810 as cystic oxide, later corrected to cystine. Despite this long history, however, a fundamental understanding of the stages of stone formation and the rational design of therapies for stone prevention have remained elusive.This Account reviews discoveries and advances from our laboratories that have unraveled the complex crystal growth mechanisms of l-cystine, which forms l-cystine kidney stones in at least 20â¯000 individuals in the U.S. alone. Although l-cystine stones affect fewer individuals than common calcium oxalate stones, they are usually larger, recur more frequently, and are more likely to cause chronic kidney disease. Real-time in situ atomic force microscopy (AFM) reveals that the crystal growth of hexagonal l-cystine is characterized by a complex mechanism in which six interlaced anisotropic spirals grow synchronously, emanating from a single screw dislocation to generate a micromorphology with the appearance of stacked hexagonal islands. In contrast, proximal heterochiral dislocations produce features that appear to be spirals but actually are closed loops, akin to a Frank-Read source. These unusual and aesthetic growth patterns can be explained by the coincidence of the dislocation Burgers vector and the crystallographic 61 screw axis. Inhibiting l-cystine crystal growth is key to preventing stone formation. Decades of studies of "tailor-made additives", which are imposter molecules that closely resemble the solute and bind to crystal faces through molecular recognition, have demonstrated their effects on crystal properties such as morphology and polymorphism. The ability to visualize crystal growth in real time by AFM enables quantitative measurements of step velocities and, by extension, the effect of prospective inhibitors on growth rates, which can then be used to deduce inhibition mechanisms. Investigations with a wide range of prospective inhibitors revealed the importance of precise molecular recognition for binding l-cystine imposters to crystal sites, which results in step pinning and the inhibition of step advancement as well as the growth of bulk crystals. Moreover, select inhibitors of crystal growth, measured in vitro, reduce or eliminate stone formation in knockout mouse models of cystinuria, promising a new pathway to l-cystine stone prevention. These observations have wide-ranging implications for the design of therapies based on tailor-made additives for diseases associated with aberrant crystallization, from disease-related stones to "xenostones" that form in vivo because of the crystallization of low-solubility therapeutic agents such as antiretroviral agents.
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Cistinúria , Cálculos Renais , Animais , Cristalização , Cistina/química , Cistina/metabolismo , Cistina/uso terapêutico , Cistinúria/complicações , Cistinúria/tratamento farmacológico , Cistinúria/metabolismo , Rim , Cálculos Renais/química , Cálculos Renais/etiologia , Cálculos Renais/prevenção & controle , Masculino , CamundongosRESUMO
BACKGROUND: Controlling malaria-transmitting Anopheles mosquitoes with pyrethroid insecticides is becoming increasingly challenging because of widespread resistance amongst vector populations. The development of new insecticides and insecticidal formulations is time consuming and costly, however. A more active crystalline form of deltamethrin, prepared by heating the commercial crystalline form, previously was reported to be 12-times faster acting against susceptible North American Anopheles quadrimaculatus mosquitoes. Herein the potential for heat-activated deltamethrin dispersed on chalk to overcome various resistance mechanisms amongst five West African Anopheles strains is investigated, and its long-term sustained lethality evaluated. METHODS: The more active deltamethrin form was generated in a commercial dust containing deltamethrin by heating the material as purchased. Tarsal contact bioassays were conducted to investigate its efficacy, potency, and speed of action against resistant Anopheles populations compared to the commercially available form of deltamethrin dust. RESULTS: In all cases, D-Fense Dust heated to generate the more active form of deltamethrin was substantially more effective than the commercially available formulation. 100% of both Banfora M and Kisumu populations were knocked down 10 min post-exposure with no recovery afterwards. Gaoua-ara and Tiefora strains exhibited 100% knockdown within 15 min, and the VK7 2014 strain exhibited 100% knockdown within 20 min. In all cases, 100% mortality was observed 24 h post-exposure. Conversely, the commercial formulation (unheated) resulted in less than 4% mortality amongst VK7 2014, Banfora, and Gaoua-ara populations by 24 h, and Tiefora and Kisumu mosquitoes experienced 14 and 47% mortality by 24 h, respectively. The heat-activated dust maintained comparable efficacy 13 months after heating. CONCLUSIONS: The heat-activated form of commercial deltamethrin D-Fense Dust outperformed the material as purchased, dramatically increasing efficacy against all tested pyrethroid-resistant strains. This increase in lethality was retained for 13 months of storage under ambient conditions in the laboratory. Higher energy forms of commonly used insecticides may be employed to overcome various resistance mechanisms seen in African Anopheles mosquitoes through more rapid uptake of insecticide molecules from their respective solid surfaces. That is, resistant mosquitoes can be killed with an insecticide to which they are resistant without altering the molecular composition of the insecticide.
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Anopheles , Inseticidas , Piretrinas , Animais , Inseticidas/farmacologia , Resistência a Inseticidas , Controle de Mosquitos/métodos , Mosquitos Vetores , Piretrinas/farmacologia , Nitrilas/farmacologiaRESUMO
Nucleoside and nucleobase analogs capable of interfering with nucleic acid synthesis have played essential roles in fighting infectious diseases. However, many of these agents are associated with important and potentially lethal off-target intracellular effects that limit their use. Based on the previous discovery of base-modified 2'-deoxyuridines, which showed high anticancer activity while exhibiting lower toxicity toward rapidly dividing normal human cells compared to antimetabolite chemotherapeutics, we hypothesized that a similar modification of the N4-hydroxycytidine (NHC) molecule would provide novel antiviral compounds with diminished side effects. This presumption is due to the substantial structural difference with natural cytidine leading to less recognizability by host cell enzymes. Among the 42 antimetabolite species that have been synthesized and screened against VEEV, one hit compound was identified. The structural features of the modifying moiety were similar to those of the anticancer lead 2'-deoxyuridine derivative reported previously, providing an opportunity to pursue further structure-activity relationship (SAR) studies directed to lead improvement, and obtain insight into the mechanism of action, which can lead to identifying drug candidates against a broad spectrum of RNA viral infections.
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Vírus da Encefalite Equina Venezuelana , Animais , Humanos , Antimetabólitos , Antivirais/farmacologia , Desoxiuridina , Cavalos , ImunossupressoresRESUMO
In this study, we report the synthesis and characterization of some heteroleptic Cr(III) complexes of the form [Cr(Phen)2L](OTf)3, where Phen = 1,10-phenanthroline and L is either 2,2'-bipyridine (bpy) or its derivatives, such as 4,4'-dimethyl-2,2'-bipyridine (4,4'-DMB), 4,4'-dimethoxy-2,2'-bipyridine (4,4'-DMOB), 4,4'-ditert-butyl-2,2'-bipyridine (4,4'-dtbpy), 5,5'-dimethyl-2,2'-bipyridine (5,5'-DMB), 4,4'-dimethoxycarbonyl-2,2'-bipyridine (4,4'-dmcbpy) or 1,10-phenanthroline derivatives, such as 5-methyl-1,10-phenanthroline (5-Me-Phen) and 4,7-dimethyl-1,10-phenanthroline (4,7-DMP). Heteroleptic complexes were prepared in two stages via the intermediate [Cr(Phen)2(CF3SO3)2](CF3SO3) and five examples have been crystallographically characterized. Steady-state absorption and luminescence emission characteristics of these complexes were measured in 1 M HCl solutions. The luminescence quantum yield of these complexes was found to be the lowest for [Cr(Phen)2(4,4'-dmcbpy)](OTf)3 and the highest for [Cr(Phen)2(4,4'-DMB)](OTf)3 with values of 0.31 × 10-2 and 1.48 × 10-2, respectively. The calculated excited state energy, E0-0, was found to vary within the narrow range of 163.1-165.0 kJ mol-1 across the series. Transient absorption spectra in degassed, air-equilibrated, and oxygen-saturated 1 M HCl aqueous solutions were also measured at different time decays and demonstrated no significant differences, indicating the absence of any ion-separated species in the excited state. Excited-state decay traces at the wavelength of maximum absorption were used to calculate oxygen quenching rate constants, kq, which were found to be in the range 3.26-5.27 × 107 M-1 s-1. Singlet oxygen luminescence photosensitized by these complexes was observed in D2O, and its luminescence intensity at 1270 nm was used for the determination of singlet oxygen quantum yields for these complexes, which were in the range of 0.20-0.44, while the fraction of the excited 2E state quenched by oxygen was in the range of 0.22-0.68, and the efficiency of singlet oxygen production was in the range of 0.44-0.90. The mechanism by which the excited 2E state is quenched by oxygen is explained by a spin statistical model that predicts the balance between charge transfer and noncharge transfer deactivation pathways, which was represented by the parameter pCT that was found to vary from 0.35 to 0.68 for this series of Cr(III) complexes.
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Pyrethroid contact insecticides are mainstays of malaria control, but their efficacies are declining due to widespread insecticide resistance in Anopheles mosquito populations, a major public health challenge. Several strategies have been proposed to overcome this challenge, including insecticides with new modes of action. New insecticides, however, can be expensive to implement in low-income countries. Here, we report a simple and inexpensive method to improve the efficacy of deltamethrin, the most active and most commonly used pyrethroid, by more than 10 times against Anopheles mosquitoes. Upon heating for only a few minutes, the commercially available deltamethrin crystals, form I, melt and crystallize upon cooling into a polymorph, form II, which is much faster acting against fruit flies and mosquitoes. Epidemiological modeling suggests that the use of form II in indoor residual spraying in place of form I would significantly suppress malaria transmission, even in the presence of high levels of resistance. The simple preparation of form II, coupled with its kinetic stability and markedly higher efficacy, argues that form II can provide a powerful, timely, and affordable malaria control solution for low-income countries that are losing protection in the face of worldwide pyrethroid resistance.
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Anopheles/efeitos dos fármacos , Inseticidas/farmacologia , Malária/prevenção & controle , Controle de Mosquitos/métodos , Nitrilas/farmacologia , Piretrinas/farmacologia , Animais , Cristalização , Drosophila melanogaster/efeitos dos fármacos , Feminino , Humanos , Resistência a Inseticidas , Inseticidas/química , Modelos Biológicos , Nitrilas/química , Piretrinas/químicaRESUMO
A synthetic strategy for fabricating colloidal particles with spatially segregated amine-functionalized lobes enables regioselective coating with gold to afford metallodielectric particles with a variety of shapes and lobe sizes. This approach can produce either dissymmetric dumbbell-shaped two-lobed Au-TPM particles (Au-T) or dissymmetric or symmetric three-lobed particles with gold coating on one (Au-T-T and T-Au-T) or two lobes (Au-T-Au). Dielectrophoretic (DEP) forces exerted by an AC field confined between two opposing electrodes generate aggregates ranging from 1D chains to 2D close-packed lattices, depending on the particle shape and lobe arrangement. The aggregate structures reflect the lowest energy configurations resulting from the induced dipole moments created in particle lobes within the confined electric field.
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The addition of the highly reactive reagent allylmagnesium halide to α-substituted acyclic chiral ketones proceeded with high stereoselectivity. The stereoselectivity cannot be analyzed by conventional stereochemical models because these reactions do not conform to the requirements of those models. Instead, the stereoselectivity arises from the approach of the nucleophile to the most accessible diastereofaces of the lowest-energy conformations of the ketones. High stereoselectivity is expected, and the stereochemical outcome can be predicted, with conformationally biased ketones that have sterically distinguishable diastereofaces wherein only one face is accessible for nucleophilic addition. The conformations of the ketones can be determined by a combination of computational modeling and, in some cases, structure determination by X-ray crystallography.
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Cetonas , Indicadores e Reagentes , Cetonas/química , Conformação Molecular , EstereoisomerismoRESUMO
Fast-scan cyclic voltammetry (FSCV) with micron-sized carbon sensors is a promising approach for monitoring the fast dynamics of serotonin (5-HT) neuromodulatory signals in the brain. However, sensor performance using FSCV can be compromised by complex chemical reactions associated with the reduction and oxidation of 5-HT, posing considerable challenges to detection of 5-HT in vivo. Herein we describe the use of engineered graphitic sensors to characterize the complex electrochemistry of 5-HT under a wide range of measurement conditions, with the aim of optimizing the FSCV conditions for in vivo quantitative 5-HT detection. These measurements reveal that water plays a significant role in driving side reactions during low-voltage FSCV measurements, leading to the observation of a well-defined secondary redox couple we associated with the redox reaction of tryptamine 4,5-dione. Remarkably, these side reactions can persist subsequent to the primary redox events associated with 5-HT. Furthermore, the results reveal a critical deviation from this ideal redox behavior if the FSCV anodic limit exceeds +0.8 V, which can be attributed to the generation of radical species from water oxidation. These new insights could lead to new FSCV protocols for more reliable 5-HT detection.
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Grafite , Serotonina , Eletroquímica/métodos , Carbono/química , EletrodosRESUMO
Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between 20 and 30 proteins to carry out their viral replication cycle, including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2'-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? Although experimentally derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over 1 ms of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16's conformational ensemble to activate it. Second, guided by this activation mechanism and Markov state models, we investigate whether Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV1 and MERS but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket.
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Imidacloprid, the world's leading insecticide, has been approved recently for controlling infectious disease vectors; yet, in agricultural settings, it has been implicated in the frightening decline of pollinators. This argues for strategies that sharply reduce the environmental impact of imidacloprid. When used as a contact insecticide, the effectiveness of imidacloprid relies on physical contact between its crystal surfaces and insect tarsi. Herein, seven new imidacloprid crystal polymorphs are reported, adding to two known forms. Anticipating that insect uptake of imidacloprid molecules would depend on the respective free energies of crystal polymorph surfaces, measurements of insect knockdown times for the metastable crystal forms were as much as nine times faster acting than the commercial form against Aedes, Anopheles, and Culex mosquitoes as well as Drosophila (fruit flies). These results suggest that replacement of commercially available imidacloprid crystals (a.k.a. Form I) in space-spraying with any one of three new polymorphs, Forms IV, VI, IX, would suppress vector-borne disease transmission while reducing environmental exposure and harm to nontarget organisms.
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Neonicotinoides , NitrocompostosRESUMO
For several decades, cell-mediated cytotoxicity has been measured using the 51 Cr release assay. This assay, however, has several drawbacks and flow cytometry has been used as an alternative to measure cytotoxic activity. Here, we present a quantitative method for cell-mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein-AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 h in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes, and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell-mediated lysis is measured by comparing target counts for different effector-to-target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target-killing activity might be compromised by small volume samples or low frequency of cytotoxic cells. © 2020 International Society for Advancement of Cytometry.
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Citotoxicidade Imunológica , Células Matadoras Naturais , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Corantes Fluorescentes , HumanosRESUMO
Experiments on autophoretic bimetallic nanorods propelling within a fuel of hydrogen peroxide show that tail-heavy swimmers preferentially orient upwards and ascend along inclined planes. We show that such gravitaxis is strongly facilitated by interactions with solid boundaries, allowing even ultraheavy microswimmers to climb nearly vertical surfaces. Theory and simulations show that the buoyancy or gravitational torque that tends to align the rods is reinforced by a fore-aft drag asymmetry induced by hydrodynamic interactions with the wall.
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This contribution describes a synthetic strategy for the fabrication of multicomponent colloidal "molecules" with controllable complex morphologies and compositionally distinct lobes. Using 3-(trimethoxysilyl)propyl methacrylate (TPM) as the building block, the methodology enables a scalable bulk synthesis of customized chiral colloidal particles with geometric and compositional chirality by a sequential seeded growth method. The synthetic protocol presents a versatile platform for constructing colloidal molecules with multiple components having customized shapes and functionalities, with the potential to impact the design of chromatic patchy particles, colloidal swimmers, and chiral optical materials, as well as informing programmable assembly.
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BACKGROUND: Detection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48-72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins. METHODS: A mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs. RESULTS: Using quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point. CONCLUSIONS: While mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.
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A crystallographic investigation of a series of host-guest complexes in which small-molecule organic guests occupy the central cavity of an approximately cubic M8 L12 coordination cage has revealed some unexpected behaviour. Whilst some guests form 1:1 Hâ G complexes as we have seen before, an extensive family of bicyclic guests-including some substituted coumarins and various saturated analogues-form 1:2 Hâ G2 complexes in the solid state, despite the fact that solution titrations are consistent with 1:1 complex formation, and the combined volume of the pair of guests significantly exceeds the Rebek 55±9 % packing for optimal guest binding, with packing coefficients of up to 87 %. Re-examination of solution titration data for guest binding in two cases showed that, although conventional fluorescence titrations are consistent with 1:1 binding model, alternative forms of analysis-Job plot and an NMR titration-at higher concentrations do provide evidence for 1:2 Hâ G2 complex formation. The observation of guests binding in pairs in some cases opens new possibilities for altered reactivity of bound guests, and also highlights the recently articulated difficulties associated with determining stoichiometry of supramolecular complexes in solution.
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The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).
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Invited for the cover of this issue is the group of Michaelâ D. Ward at the University of Warwick. The image depicts structures of the host cage containing one guest or two guests. Read the full text of the article at 10.1002/chem.201905499.