Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC₅₀?=?260?±?0.112?µg/ml) and antiplasmodial (IC₅₀?=?8.5?±?1.6?µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31?µg/ml?≥?IC₅₀?≥?5.6?µg/ml; antiplasmodial: 14?µg/ml?≥?IC₅₀?≥?2?µg/ml) with very minimal toxicity.     

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

1. Download high-res image (183KB)
2. Download full-size image

     

Functioning of plant-bacterial associations under osmotic stress in vitro

World Journal of Microbiology and Biotechnology - Derived from RNA, 5?-ribonucleotides, especially Inosine-5?-monophosphate (IMP) and guanosine-5?-monophosphate (GMP), can enhance...     

Autoinhibition Mechanism of the Ubiquitin-Conjugating Enzyme UBE2S by Autoubiquitination

1. Download : Download high-res image (158KB)
2. Download : Download full-size image

     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.     

Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.     

B3LYP/6-311++G* * study of structure and spin-spin coupling constant in heparin disaccharide

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.