Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Biosynthesis of Al2O3 and europium-doped Al2O3 nanoparticle using Cymbopogon citratus (lemongrass) extract and its antibacterial property

Cymbopogon citratus-mediated pure aluminium oxide (Al₂O₃) and europium (Eu)-doped Al₂O₃ with different amounts of metal ion were prepared using a green synthesis method. Synthesised nanoparticles were characterised by ultraviolet (UV)-visible spectroscopy, photoluminescence (PL), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). Synthesis of nanoparticles is confirmed by using UV-visible spectroscopy showing maximum absorption at 411 and 345 nm for Al₂O₃ and Eu-doped Al₂O₃, respectively. The antibacterial activity of prepared nanoparticles was evaluated against Pseudomonas aeruginosa, Streptococcus aureus, Escherichia coli and Klebsiella pneumoniae using a well-diffusion technique. The effect of pure Al₂O₃ and Eu-doped nanoparticles shows excellent results against P. aeruginosa, S. aureus, E. coli and K. pneumoniae.     

Activity of Pseudomonas aeruginosa in biofilms: effect of calcium

Aerobic glucose metabolism by Pseudomonas aeruginosa biofilms at various calcium loading rates was investigated. The influence of calcium on specific growth rate, extracellular polymeric substance (EPS) formation rate, biofilm detachment rate, and biofilm calcium concentrations was determined. Calcium accumulated in the biofilm in proportion to the liquid phase concentration. Increasing calcium concentration increased the cohesiveness of the biofilm as indicated by a lower relative detachment rate. Specific activity in the biofilm was the same as that measured in a chemostat and was not influenced by changing calcium concentration. EPS formation rate in the biofilm was unaffected by calcium concentration but was higher than that observed in a chemostat.     

Study on luminescence properties of Ce3+ and Eu3+ ions in a nanocrystalline hexagonal Zn4Al22O37 novel system

The present communication is strongly focused on the investigation of synthesis, structural and luminescence properties of cerium (Ce³⁺)- and europium (Eu³⁺)-activated Zn₄Al₂₂O₃₇ phosphors. Ce³⁺- and Eu³⁺-doped Zn₄Al₂₂O₃₇ novel phosphors were prepared using a solution combustion synthesis route. Structural properties were studied using powder X-ray diffraction and high-resolution transverse electron microscopy. The optical properties were studied using ultraviolet–visible light spectroscopy and Fourier transform infrared spectroscopy; luminescence properties were studied using a photoluminescence (PL) technique. The crystal structure of the prepared Zn₄Al₂₂O₃₇ host and Ce³⁺- and Eu³⁺-activated Zn₄Al₂₂O₃₇ phosphors was investigated and was found to have a hexagonal structure. The measured PL emission spectrum of the Ce³⁺-doped Zn₄Al₂₂O₃₇ phosphor showed an intense and broad emission band centred at 421 nm under a 298 nm excitation wavelength. By contrast, the Eu³⁺-doped Zn₄Al₂₂O₃₇ phosphor exhibited two strong and intense emission bands at approximately 594 nm (orange) and 614 nm (red), which were monitored under 395 nm excitation. The Commission Internationale de l’Eclairage (CIE) colour coordinates of the Ce³⁺-doped Zn₄Al₂₂O₃₇ were investigated and found to be x = 0.1567, y = 0.0637 (blue) at 421 nm and for Eu³⁺-doped Zn₄Al₂₂O₃₇ were x = 0.6018, y = 0.3976 (orange) at 594 nm and x = 0.6779, y = 0.3219 (red) at 614 nm emission. The luminescence behaviour of the synthesized phosphors suggested that these phosphors may be used in lighting applications.     

EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.     

phastSim: Efficient simulation of sequence evolution for pandemic-scale datasets

Sequence simulators are fundamental tools in bioinformatics, as they allow us to test data processing and inference tools, and are an essential component of some inference methods. The ongoing surge in available sequence data is however testing the limits of our bioinformatics software. One example is the large number of SARS-CoV-2 genomes available, which are beyond the processing power of many methods, and simulating such large datasets is also proving difficult. Here, we present a new algorithm and software for efficiently simulating sequence evolution along extremely large trees (e.g. > 100, 000 tips) when the branches of the tree are short, as is typical in genomic epidemiology. Our algorithm is based on the Gillespie approach, and it implements an efficient multi-layered search tree structure that provides high computational efficiency by taking advantage of the fact that only a small proportion of the genome is likely to mutate at each branch of the considered phylogeny. Our open source software allows easy integration with other Python packages as well as a variety of evolutionary models, including indel models and new hypermutability models that we developed to more realistically represent SARS-CoV-2 genome evolution.     

Phospholipase C epsilon suppresses integrin activation

Phospholipase Cepsilon (PLCepsilon) is a newly described effector of the small GTP-binding protein H-Ras. Utilizing H-Ras effector mutants, we show that mutants H-Ras(G12V/E37G) and H-Ras(G12V/D38N) suppressed integrin activation in an ERK-independent manner. H-Ras(G12V/D38N) specifically activated the PLCepsilon effector pathway and suppressed integrin activation. Inhibition of PLCepsilon activation with a kinase-dead PLCepsilon mutant prevented H-Ras(G12V/D38N) from suppressing integrin activation, and low level expression of H-Ras(G12V/D38N) could synergize with wild-type PLCepsilon to suppress integrins. In addition, knockdown of endogenous PLCepsilon with small interfering RNA blocked H-Ras(G12V/D38N)-mediated integrin suppression. Suppressing integrin function with the H-Ras(G12V/D38N) mutant reduced cell adhesion to von Willebrand factor and fibronectin; this reduction in cell adhesion was blocked by coexpression of the kinase-dead PLCepsilon mutant. These results show that H-Ras suppresses integrin affinity via independent Raf and PLCepsilon signaling pathways and demonstrate a new physiological function for PLCepsilon in the regulation of integrin activation.