Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

Superiority of Classification Tree versus Cluster,Fuzzy and Discriminant Models in a Heartbeat Classification System

This study presents a 2-stage heartbeat classifier of supraventricular (SVB) and ventricular (VB) beats. Stage 1 makes computationally-efficient classification of SVB-beats, using simple correlation threshold criterion for finding close match with a predominant normal (reference) beat template. The non-matched beats are next subjected to measurement of 20 basic features, tracking the beat and reference template morphology and RR-variability for subsequent refined classification in SVB or VB-class by Stage 2. Four linear classifiers are compared: cluster, fuzzy, linear discriminant analysis (LDA) and classification tree (CT), all subjected to iterative training for selection of the optimal feature space among extended 210-sized set, embodying interactive second-order effects between 20 independent features. The optimization process minimizes at equal weight the false positives in SVB-class and false negatives in VB-class. The training with European ST-T, AHA, MIT-BIH Supraventricular Arrhythmia databases found the best performance settings of all classification models: Cluster (30 features), Fuzzy (72 features), LDA (142 coefficients), CT (221 decision nodes) with top-3 best scored features: normalized current RR-interval, higher/lower frequency content ratio, beat-to-template correlation. Unbiased test-validation with MIT-BIH Arrhythmia database rates the classifiers in descending order of their specificity for SVB-class: CT (99.9%), LDA (99.6%), Cluster (99.5%), Fuzzy (99.4%); sensitivity for ventricular ectopic beats as part from VB-class (commonly reported in published beat-classification studies): CT (96.7%), Fuzzy (94.4%), LDA (94.2%), Cluster (92.4%); positive predictivity: CT (99.2%), Cluster (93.6%), LDA (93.0%), Fuzzy (92.4%). CT has superior accuracy by 0.3–6.8% points, with the advantage for easy model complexity configuration by pruning the tree consisted of easy interpretable ‘if-then’ rules.     

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.     

Studies on the antioxidant activity of some chromonylrhodanine derivatives

Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2‐diphenyl‐1‐picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric‐ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin‐trapping technique. The 5,5‐dimethyl‐ 1‐pyrroline‐1‐oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO‐OOH radical EPR signal and the DMPO‐OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC₅₀: 0.51–0.56 µmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd.     

High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Iα subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23±0.04 vs. 0.54±0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGIα dimerization.     

Siroheme: An essential component for life on earth

Life on earth is dependent on sulphur (S) and nitrogen (N). In plants, the second step in the reduction of sulphate and nitrate are mediated by the enzymes sulphite and nitrite reductases, which contain the iron (Fe)-containing siroheme as a cofactor. It is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in the plastids via three enzymatic reactions, methylation, oxidation and ferrochelatation. Without siroheme biosynthesis, there would be no life on earth. Limitations in siroheme should have an enormous effect on the S- and Nmetabolism, plant growth, development, fitness and reproduction, biotic and abiotic stresses including growth under S, N and Fe limitations, and the response to pathogens and beneficial interaction partners. Furthermore, the vast majority of redoxreactions in plants depend on S-components, and S-containing compounds are also involved in the detoxification of heavy metals and other chemical toxins. Disturbance of siroheme biosynthesis may cause the accumulation of light-sensitive intermediates and reactive oxygen species, which are harmful, or they can function as signaling molecules and participate in interorganellar signaling processes. This review highlights the role of siroheme in these scenarios.Key words: iron, nitrogen, plant/microbe interaction, redox, siroheme, sulphur     

Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site.     

Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome

Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.Prokaryotes are highly diverse (33). One of the explanations of this diversity is the high extinction rate, due to genetic aggression, which leads to the clearance of ecological niches and, as a result, may allow new prokaryotic species to emerge. In the absence of host defense, viral infection of prokaryotic colonies results in colony extinction or the fixation of a fraction of the invader''s genetic material in the host genome, profoundly affecting the life cycle of the host (32). Thus, bacteria and archaea have developed various kinds of defense mechanisms to resist this pressure; the best studied of these mechanisms is restriction-modification systems (4).Along with well-known prokaryotic defense mechanisms, such as rapid evolution of cell receptors or the use of restriction-modification or toxin-antitoxin systems (see, e.g., references 6, 21, and 25), newly discovered clustered regularly interspaced palindromic repeat (CRISPR) systems seem to play an important role in protecting the cell from archaeal virus or bacteriophage assaults (reviewed in reference 36). A typical CRISPR system is a genetic locus comprising CRISPR-associated (cas) genes coding for proteins of several distinct functional classes (8, 19, 29) and a CRISPR cassette. A CRISPR cassette is formed by almost identical direct repeats with an average length of 32 nucleotides (nt), which are separated by similarly sized, unique spacers. A considerable proportion of spacers is similar to known phage or virus sequences, suggesting that the system is involved in antivirus defense (8, 29, 31). This involvement was experimentally demonstrated when a CRISPR system was shown to be essential for cell survival after invasion by foreign DNA (5). The mechanism is thought to be analogous to eukaryotic RNA interference (29), but it has not been characterized in detail yet.CRISPR cassettes retain information that could be used to reveal the evolutionary history of individual systems. First, it has been shown that CRISPR-associated genes could be divided into eight subtypes according to operon organization and gene phylogeny (19). Second, the repeats of different CRISPR cassettes may be similar, which might indicate a common origin of such cassettes. The first attempt to cluster CRISPR cassettes by the similarity of repeat sequences resulted in 12 clusters (27). In that study, the cassettes were obtained by the application of PILER-CR to completely sequenced genomes. Third, pairwise comparison of spacers could also reveal the specific evolutionary history of individual CRISPR cassettes.So far, most large-scale studies of CRISPR systems have been restricted to well-studied organisms with completely sequenced genomes (5, 9, 20, 28, 30). However, the dynamic interaction between viruses or phages and microorganisms in natural environments is of particular interest (2, 10, 15, 23, 35, 38, 40-42). It may be studied using CRISPRs in a metagenome, that is, sequenced DNA fragments collected in one geographical location and therefore representing one ecological niche with all its inhabitants. This approach is interesting for two reasons. First, metagenomic samples provide a common census of coexisting organisms, i.e., in many cases, both the infecting viruses and phages and their victims. Second, most bacteria and archaea from metagenomic samples cannot be cultivated, and hence little is known about their CRISPR systems.To date, three studies have considered host-virus interactions in metagenomes. One study used two thermophilic Synechococcus isolates from microbial mats in hot springs at Yellowstone National Park to demonstrate fast coevolution of the host and phage genomes (22). Two studies described archaeal and bacterial interactions with viruses and phages, respectively, in acidophilic biofilms (2, 39). All environmental communities analyzed so far are extreme and are dominated by few species. Natural samples containing many diverse coexisting organisms may arguably be more interesting.The largest available metagenome, produced by the Sorcerer II Global Ocean Sampling (GOS) expedition, comprises samples of genetic material collected from more than 50 geographical locations of the Pacific and Atlantic oceans (34). This variety provides an opportunity to study the evolution of phage-host interactions reflected in CRISPRs.Three algorithms, PILER-CR (14), the CRISPR recognition tool (CRT) (7), and CRISPRFinder (18), have been developed as tools for the discovery of new CRISPR cassettes. All these algorithms define candidate CRISPR cassette sequences as short direct repeats separated by short unique spacers; they then use a variety of standard repeat-finding techniques. However, the implementation of specific details is different.PILER-CR constructs local alignments of the input sequence to itself; each hit between two close regions is a candidate for an alignment of a repeat with its neighbor copy. In terms of dynamic programming, taking into account the repeat structure of a CRISPR cassette implies looking for hits only within a relatively narrow band around the main diagonal of the dot plot. This process is followed by several refinement steps.CRT does not use alignments to identify candidate repeats; rather, it derives them directly from the analysis of an input sequence. It is based on finding series of short repeats of a specified length (searching for exact k-mer matches) and then extending these repeats (increasing k-mer length) while allowing for a certain level of mismatches.Finally, CRISPRFinder is based on a suffix-tree-based algorithm for repeat discovery, again with additional refinement.All three algorithms were used for the CRISPR cassette search in this study.