Molecular Biological Background of the Species and Organ Specificity of Influenza A Viruses

Influenza is one of the great plagues which is not yet under control. The reason for this is the immense variability of the infecting agent, the influenza A viruses. These viruses behave like a chameleon: they adapt very rapidly to varying environments. New strains are “synthesized,” which can escape the immune response of the host, cross species barriers, and become highly pathogenic. We are beginning to understand the molecular background of this extraordinarily high variability. The genome of influenza A viruses consists of eight single-stranded RNA segments, each of which constitutes a gene. The total base sequence of the eight RNA segments of several strains is known. If a suitable organism becomes doubly infected with two different influenza A strains, each of the RNA segments behaves like a chromosome. This means that by reassortment of the 16 RNA segments, 2⁸ ?2 = 254 new combinations (= reassortants) are theoretically possible, each having different properties. Furthermore, mutations in the various RNA segments are relatively easily tolerated. Another great problem resides in the enormous reservoir of different influenza A viruses in the animal kingdom, especially in feral waterbirds. In these birds the avian influenza A viruses normally cause at most mild symptoms, and therefore these viruses are distributed over and between continents. Bearing this in mind it appears to be necessary to develop new ideas as to how to overcome this great plague.     

Targeted and Specific Modification with 4-Carboxybenzeneboronic Acid and TPGS of Fluorescent Gold Nanoparticles and the Enhanced Antitumor Activity Research

A targeting and specific fluorescent gold nanoparticle is prepared for tracing of drug and effective treatment of cancers. First, 4-carboxybenzeneboronic acid (CBPA) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) are used to modify cyclodextrins (CDs), then modified cyclodextrins and polyethylene glycol (PEG) are used to stabilize gold nanoclusters to prepare fluorescent gold nanoparticles (AuNCs@CD-TPGS-CBPA/PEG). CBPA has targeting and TPGS can induce apoptosis, therefore, AuNCs@CD-TPGS-CBPA/PEG has targeting and certain anti-tumor activity. The structure and morphology of the nanomaterial are characterized by using nuclear magnetic resonance (NMR) spectroscopy and transmission electron microscope (TEM). Paclitaxel (PTX) is loaded into the hydrophobic cavity of CDs to form targeting specific drug-loaded fluorescent gold nanoparticles (AuNCs@CD-TPGS-CBPA/PEG/PTX). Biological performances of AuNCs@CD-TPGS-CBPA/PEG are studied by in vitro and in vivo experiments. In vitro test results confirm that AuNCs@CD-TPGS-CBPA/PEG could be internalized by tumor cells, and effectively against tumor cells, but it is biocompatible to normal cells. In vivo experiments prove that AuNCs@CD-TPGS-CBPA/PEG has excellent targeting, biocompatibility, and enhanced antitumor capability, could enrich and stay for a long time in the tumor regions, effectively prolong the lifetime of tumor-bearing mice. Therefore, this work provides an insight into the development of fluorescent gold nanomaterials for practical biomedical application.     

Development and Validation of 2-Azaspiro [4,5] Decan-3-One (Impurity A) in Gabapentin Determination Method Using qNMR Spectroscopy

The authors developed a ¹H qNMR test procedure for identification and quantification of impurity A present in gabapentin active pharmaceutical ingredient (API) and gabapentin products. The validation studies helped to determine the limit of quantitation and assess linearity, accuracy, repeatability, intermediate precision, specificity, and robustness of the procedure. Spike-and-recovery assays were used to calculate standard deviations, coefficients of variation, confidence intervals, bias, Fisher’s F test, and Student’s t-test for assay results. The obtained statistical values satisfy the acceptance criteria for the validation parameters. The authors compared the results of impurity A quantification in gabapentin APIs and capsules by using the ¹H qNMR and HPLC test methods.     

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine, respectively. Both strains utilized all NAPPs tested as the sole source of carbon, nitrogen and energy for growth. Mineralization of NAPPs was independent of the alkyl chain length and the size of the polyamine moiety. NAPPs degraded in closed bottle tests (CBTs) using both river water and activated sludge. However, ready biodegradability of NAPPs with alkyl chain lengths of 16–18 carbon atoms and polyamine moieties with three and four nitrogen atoms could not be demonstrated. Biodegradation in the CBT was hampered by their limited bioavailability, making assessment of the true ready biodegradability of these highly adsorptive surfactants impossible. All NAPPs are therefore classified as readily biodegradable through read-across. Read-across is justified by the broad substrate specificity of NAPP-degrading microorganisms, their omnipresence and the mineralization of NAPPs.     

Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate‐Utilizing Enzymes

Chorismate‐utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free‐living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.     

A component analysis of the free energies of folding of 35 proteins: A consensus view on the thermodynamics of folding at the molecular level

What factors favor protein folding? This is a textbook question. Parsing the experimental free energies of folding/unfolding into diverse enthalpic and entropic components of solute and solvent favoring or disfavoring folding is not an easy task. In this study, we present a computational protocol for estimating the free energy contributors to protein folding semi‐quantitatively using ensembles of unfolded and native states generated via molecular dynamics simulations. We tested the methodology on 35 proteins with diverse structural motifs and sizes and found that the calculated free energies correlate well with experiment (correlation coefficient ∼ 0.85), enabling us to develop a consensus view of the energetics of folding. As a more sensitive test of the methodology, we also investigated the free energies of folding of an additional 33 single point mutants and obtained a correlation coefficient of 0.8. A notable observation is that the folding free energy components appear to carry signatures of the fold (SCOP classification) of the protein. © 2017 Wiley Periodicals, Inc.     

Candida antarctica lipase B catalysed kinetic resolution of 1,2,3,4-tetrahydro-ß-carbolines: Substrate specificity

In the frame of substrate specificity, CAL-B-catalysed asymmetric N-alkoxycarbonylations of 1-substituted tetrahydro-ß-carbolines (Me, Et, Pr, iPr) have been studied. High enantioselectivities (>200) were observed, when alkoxycarbonylation of racemic compounds (±)-1,3,5,7 were performed in DIPE in the presence of phenyl allyl carbonate and Et₃N at 60?°C using ultrasound shaking method. The reaction time increased considerably with increasing substituent size on C1; however, the isopropyl-substituted compound proved to be too bulky for the optimum activity of CAL-B. The (R)-carbamate enantiomers were hydrolysed using Pd₂(dba)₃.CHCl₃ and the enantiomers of the free amines were obtained with excellent ee (>99%).     

Substrate specificity and reaction mechanism of recombinant styrene oxide isomerase from Pseudomonas putida S12

To clarify the substrate specificity of the recombinant styrene oxide isomerase, various epoxides were subjected to the reaction. From the substituent effect on the rate of isomerization, the mechanism of the isomerase catalyzed reaction was estimated.     

Effect of single active-site cleft mutation on product specificity in a thermostable bacterial cellulase

Mutation of a single active-site cleft tyrosyl residue to a glycyl residue significantly changes the mixture of products released from phosphoric acidswollen cellulose (PSC) by EIcd, the catalytic domain of the endoglucanase-I from Acidothermus cellulolyticus. The percentage of glucose in the product stream is almost 40% greater for the Y245G mutant (and for an additional double mutant, Y245G/Q204A) than for the wild type enzyme. Comparisons of results for digestion PSC and of pretreated yellow poplar suggest that the observed shifts in product specificity are connected to the hydrolysis of a more easily digestible fraction of both substrates. A model is presented that relates the changes in product specificity to a mutation-driven shift in indexing of the polymeric substrate along the extended binding-site cleft.