Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.     

Insight of smart biosensors for COVID-19: A review

The review discusses the diagnostic application of biosensors as point-of-care devices in the COVID-19 pandemic. Biosensors are important analytical tools that can be used for the robust and effective detection of infectious diseases in real-time. In this current scenario, the utilization of smart, efficient biosensors for COVID-19 detection is increasing and we have included a few smart biosensors such as smart and intelligent based biosensors, plasmonic biosensors, field effect transistor (FET) biosensors, smart optical biosensors, surface enhanced Raman scattering (SERS) biosensor, screen printed electrode (SPE)-based biosensor, molecular imprinted polymer (MIP)-based biosensor, MXene-based biosensor and metal–organic frame smart sensor. Their significance as well as the benefits and drawbacks of each kind of smart sensor are mentioned in depth. Furthermore, we have compiled a list of various biosensors which have been developed across the globe for COVID-19 and have shown promise as commercial detection devices. Significant challenges in the development of effective diagnostic methods are discussed and recommendations have been made for better diagnostic outcomes to manage the ongoing pandemic effectively.     

Chloramine T-mediated radioiodination of proteins on intact polyacrylamide slab gels

A simple, inexpensive method is described here for the radioiodination of proteins on intact, developed polyacrylamide gels. The method is based on the chloramine T iodination procedure which yields proteins containing ¹²⁵I label specifically in tyrosine residues. When employed with intact polyacrylamide gels, our method allows detection of proteins in amounts too small to be observed by chemical stains, including Coomassie blue. The procedure should, therefore, be useful for analyzing protein mixtures where only a small amount of material is available or for assaying trace contaminants in purified protein preparations. Proteins radioactively labeled by our method are suitable for further analysis by proteolytic cleavage followed by peptide mapping or “fingerprinting.”     

High-resolution genotyping of Pseudomonas aeruginosa strains linked to acute post cataract surgery endophthalmitis outbreaks in India

Background

The number of Salmonella strains with reduced susceptibility to fluoroquinolones has increased during recent years in many countries, threatening the value of this antimicrobial group in the treatment of severe salmonella infections.

Methods

We analyzed the in vitro activities of ciprofloxacin and 10 additional fluoroquinolones against 816 Salmonella strains collected from Finnish patients between 1995 and 2003. Special attention was focused on the efficacy of newer fluoroquinolones against the Salmonella strains with reduced ciprofloxacin susceptibility.

Results

The isolates represented 119 different serotypes. Of all 816 Salmonella strains, 3 (0.4%) were resistant to ciprofloxacin (MIC ≥ 4 μg/ml), 232 (28.4%) showed reduced susceptibility to ciprofloxacin (MIC ≥ 0.125 – 2 μg/ml), and 581 (71.2%) were ciprofloxacin-susceptible. The MIC₅₀ and MIC₉₀ values of ciprofloxacin for these strains were 0.032 and 0.25 μg/ml, respectively, being lower than those of the other fluoroquinolone compounds presently on market in Finland (ofloxacin, norfloxacin, levofloxacin, and moxifloxacin). For two newer quinolones, clinafloxacin and sitafloxacin, the MIC₅₀ and MIC₉₀ values were lowest, both 0.016 and 0.064 μg/ml, respectively. Moreover, clinafloxacin and sitafloxacin exhibited the lowest MIC₅₀ and MIC₉₀ values, 0.064 and 0.125 μg/ml, against the 235 Salmonella strains with reduced susceptibility and strains fully resistant to ciprofloxacin.

Conclusion

Among the registered fluoroquinolones in Finland, ciprofloxacin still appears to be the most effective drug for the treatment salmonella infections. Among the newer preparations, both clinafloxacin and sitafloxacin are promising based on in vitro studies, especially for strains showing reduced ciprofloxacin susceptibility. Their efficacy, however, has not been demonstrated in clinical investigations.     

Evaluation of polymerized rosin for the formulation and development of transdermal drug delivery system: A technical note

Conclusions  Results from the present study conclude that PR in combination with PVP and with incorporation of dibutyl phthalate (30% wt/wt) produces smooth flexible films with improved tensile strength and percentage elongation. The release rate of drug from films and permeation across skin increases with increase in drug and PVP loading but is independent of film thickness. Patches containing PR:PVP (7:3) show promise for pharmacokinetic and pharmacodynamic performance evaluation in a suitable animal model. In view of the overall results reported in the present study, it may be proposed that PR can be used in the design of a matrix type transdermal drug delivery system to prolong the drug release. Published: December 27, 2005     

Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.     

The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis

Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.     

Tyrphostins are inhibitors of guanylyl and adenylyl cyclases

Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues. The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain. Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD). In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro. Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM. The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor. Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM. As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP. Inhibition was reversible, ruling out a covalent modification of the receptor. Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25. Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity. Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.     

Bufadienolides from Drimia robusta and Urginea epigea (Hyacinthaceae)

Two bufadienolides, 6beta-acetoxy-3beta,8beta,14beta-trihydroxy-12-oxobufa-4,20,22-trienolide and 14beta-hydroxybufa-3,5,20,22-tetraenolide were isolated from the dichloromethane extract of the bulbs of Drimia robusta and the methanol extract of the bulbs of Urginea epigea, respectively. The bulbs of Drimia robusta also yielded several known compounds, 6beta-acetoxy-3beta,8beta,12beta,14beta-tetrahydroxybufa-4,20,22-trienolide (12beta-hydroxyscillirosidin) from the dichloromethane extract and three common aromatic acids, 4-hydroxy-3-methoxybenzoic acid, 3,4-dihydroxybenzoic acid, and trans-3-(4'-hydroxyphenyl)-2-propenoic acid from the ethyl acetate extract.