Effective Synthesis of Fluorescently Labeled Morpholino Nucleoside Triphosphate Derivatives

Morpholino nucleoside triphosphates (A, U, G, C, T) bearing the active functional amino group tethered to morpholine residue and their fluorescently labeled derivatives were synthesized. All compounds were characterized by ¹H, ¹³C, and ³¹P NMR, and mass spectrometry. A possibility of using fluorescently labeled morpholino nucleoside triphosphates as chain terminators in DNA sequencing is discussed.     

A New Mouse Model of Mild Ornithine Transcarbamylase Deficiency (spf-j) Displays Cerebral Amino Acid Perturbations at Baseline and upon Systemic Immune Activation

Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N). This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.     

Activation of Antioxidant Defenses in Whole Saliva by Psychosocial Stress Is More Manifested in Young Women than in Young Men

Psychosocial stress has been long known to have deleterious effects on health. Nevertheless, an exposure to moderate stressors enhances resilience and promotes health benefits. Male and female organisms differ in many aspects of health and disease. The aim of this study was to investigate antioxidant activity and oxidative damage in saliva in a psychosocial stress paradigm in men and women. Here, we show that an acute stressor of moderate strength augments antioxidant activity and decreases oxidative damage in whole saliva of young people. An examination stress caused a significant increase of catalase activity, accompanied by a decrease of levels of oxidized proteins. Levels of thiobarbituric acid-reacting substances did not increase at stress, indicating that lipid peroxidation was not activated. The stress-induced alterations were more manifested in young women compared to young men. Thus, antioxidant protective mechanisms are more activated by a moderate stressor in young women than in young men.     

New insights into molecular mechanism(s) underlying the presynaptic action of nitric oxide on GABA release

Background

Nitric oxide (NO) is an important presynaptic modulator of synaptic transmission. Here, we aimed to correlate the release of the major inhibitory neurotransmitter GABA with intracellular events occurring in rat brain axon terminals during their exposure to NO in the range of nanomolar–low micromolar concentrations.

Methods

Using [³H]GABA and fluorescent dyes (Fluo 4-AM, acridine orange and rhodamine 6G), the following parameters were evaluated: vesicular and cytosolic GABA pools, intracellular calcium concentration, synaptic vesicle acidification, and mitochondrial membrane potential. Diethylamine NONOate (DEA/NO) and S-nitroso-N-acetylpenicillamine (SNAP) were used as NO donors.

Results

DEA/NO and SNAP (in the presence of dithiothreitol (DTT)) stimulated external Ca^2 +-independent [³H]GABA release, which was not attributed to a rise in intracellular calcium concentration. [³H]GABA release coincided with increasing GABA level in cytosol and decreasing the vesicular GABA content available for exocytotic release. There was a strong temporal correlation between NO-induced increase in cytosolic [GABA] and dissipation of both synaptic vesicle proton gradient and mitochondrial membrane potential. Dissipation was reversible, and recovery of both parameters correlated in time with re-accumulation of [³H]GABA into synaptic vesicles. The molar ratio of DTT to SNAP determined the rate and duration of the recovery processes.

Conclusions

We suggest that NO can stimulate GABA release via GABA transporter reversal resulting from increased GABA levels in cytosol. The latter is reversible and appears to be due to S-nitrosylation of key proteins, which affect the energy status of the pre-synapse.

General significance

Our findings provide new insight into molecular mechanism(s) underlying the presynaptic action of nitric oxide on inhibitory neurotransmission.     

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.Culture-independent 16S rRNA gene surveys are now routinely utilized to examine the microbial diversity in various environmental habitats. However, in surveys of highly diverse ecosystems, the size of clone libraries typically constructed (100 to 500 clones) allows for the identification only of members of the community that are present in high abundance (2, 13, 14, 17, 24, 51). In addition to the failure to detect the rare members of the ecosystem, these relatively small datasets provide inaccurate estimates when used for computing species richness within an ecosystem. Regardless of the approach utilized to estimate species richness, the estimates obtained are highly dependent on sample size, and smaller datasets typically result in the underestimation of species richness (14, 44, 47, 55).The use of a pyrosequencing-based approach (40) in 16S gene-based diversity surveys promises to overcome both of the above-mentioned problems associated with inadequate sampling. The large number of 16S rRNA gene sequences produced (hundreds of thousands) allows access to rare members of the community (25; J. M. Tiedje, presented at the 108th General Meeting of the American Society for Microbiology, Boston, MA, 2008), as well as a relatively more accurate estimation of species richness. However, with the introduction of this new technology, it is necessary to correlate the results obtained from newer pyrosequencing-based surveys to the extensive collection of longer, capillary sequence-generated 16S rRNA gene sequences that has been deposited in public databases during the last 2 decades. Several recent studies have examined the utility of pyrosequencing fragments in providing an accurate survey of overall community structure (36) and investigated the ability of various fragments spanning the 16S rRNA gene to accurately predict the phylogenetic affiliation of pyrosequencing-generated fragments at various taxonomic cutoffs (35, 54). As such, these admirable efforts gave useful insights into the advantages and limitations of the pyrosequencing approach in 16S-based community surveys, pinpointed specific regions that provide better phylogenetic resolution than other pyrosequencing-generated regions, and provided a quantitative assessment of binning accuracy at various empirical cutoffs.However, while issues regarding correlating phylogenies of shorter and longer fragments are actively being addressed, efforts to calibrate species richness data obtained from various pyrosequencing fragments at various taxonomic cutoffs to estimates obtained using longer 16S rRNA gene fragments are still lacking. It is unclear how pairwise distances and, hence, operational taxonomic unit (OTU) assignments and species richness estimates computed using various shorter fragments spanning various regions of the 16S rRNA gene will correlate to pairwise distances computed using the nearly complete 16S rRNA gene. Elucidating such differences between shorter and nearly complete fragments, as well as between shorter fragments representing different regions in the 16S rRNA gene, is absolutely necessary for accurate meta-analysis of species richness in previously published and future datasets constructed using various sequencing approaches.Here, we constructed, sequenced, and analyzed a 16S rRNA library of 1,132 clones generated from an undisturbed tallgrass prairie soil in central Oklahoma and compared the numbers of OTUs and species richness values obtained using the full-length data sets (with and without the application of the Lane mask filter that excludes hypervariable regions from the phylogenetic analysis) (32) and fragments simulating pyrosequencing output generated by clipping where known conserved bacterial primers are encountered in the 16S rRNA gene. The lengths of the chosen simulated-pyrosequencing fragments represent amplicons that have been generated using the original GS20 pyrosequencing platform (≈100 bp) (25, 44, 48), similar to those currently being generated using the GS FLX pyrosequencing platform (≈250 bp) (1, 20, 35) or amplicons produced using the anticipated increase in the new GS XLR pyrosequencing platform (>250 bp). We show that the choice of the pyrosequenced fragment could indeed impact the number of OTUs calculated at different taxonomic cutoffs, with some fragments underestimating and others overestimating such parameters compared to the results with longer, nearly complete 16S rRNA gene fragments. We also show that even more marked differences could be encountered when comparing two pyrosequencing fragments within the same molecule. Further, we established a regression analysis that explains the nature of the observed discrepancies using the proportions of the hypervariable, variable, and conserved bases within fragments.     

Correction: Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Correction to Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial Amina Abdelaal, Hassan Abd El-Ghaffar, Mohammad Hosam Eldeen Zaghloul, Noha El mashad, Ehab Badran, Amal Fathy Annals of Clinical Microbiology and Antimicrobials 2009, 8:4 (30 January 2009)     

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory–secretory candidate vaccine antigens

Excretory–secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naïve, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P < 0.0001) in vitro attrition of lung-stage larvae. Thus, our data suggested that a balance between Th1, Th17, and Th2 cytokines is required for effective schistosome larval elimination.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

Synthesis, binding studies and molecular modeling of novel cannabinoid receptor ligands

In the present work, we report upon the design, synthesis and biological evaluation of new anandamide derivatives obtained by modifications of the fatty acyl chain and/or of the ethanolamide 'tail'. The compounds are of the general formula: 6-(substituted-phenyl)/naphthyl-4-oxohex-5-enoic acid N-substituted amide and 7-naphthyl-5-oxohept-6-enoicacid N-substituted amide. The novel compounds had been evaluated for their binding affinity to CB1/CB2 cannabinoid receptors, binding studies showed that some of the newly developed compounds have measurable affinity and selectivity for the CB2 receptor. Compounds XI and XVIII showed the highest binding affinity for CB2 receptor. None of the compounds exhibited inhibitory activity towards anandamide hydrolysis, thus arguing in favor of their enzymatic stability. The structure-activity relationship has been extensively studied through a tailor-made homological model using constrained docking in addition to pharmacophore analysis, both feature and field based.