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.     

Cytotoxic isoferulic acidamide from Myricaria germanica (Tamaricaceae)

Tamgermanitin, a unique N-trans-Isoferuloyltyramine, together with the hitherto unknown polyphenolics, 2,4-di-O-galloyl-(α/β)-glucopyranose and kaempferide 3,7-disulphate have been isolated from the leaf aqueous ethanol extract of the false tamarisk, Myricaria germanica DESV. In addition, 18 known phenolics were also separated and characterized. All structures were elucidated on the basis of detailed analysis of 1D- ¹H and ¹³C NMR, COSY, HSQC, HMBC and HRFTESIMS spectral data. The extract, its chromatographic column fractions and the isolated isoferuloyltyramine, tamgermanetin demonstrated potential cytotoxic effect against three different tumor cell lines, namely liver (Huh-7), breast (MCF-7) and prostate (PC-3). The IC₅₀''s were found to be substantially low with low-resistance possibility. DNA flow-cytometic analysis indicated that column fractions and tamgermanetin enhanced pre-G apoptotic fraction. Both materials showed inhibiting activity against PARP enzyme activity. In conclusion, we report the isolation and identification of a novel compound, tamgermanitin, from the aqueous ethanol extract of Myricaria germanica leaves. Further, different fractions of the extract and tamgermanitin exhibit potent cytotoxic activities which warrant further investigations.     

Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a K_i of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements.     

Discovery of a novel IKK-β inhibitor by ligand-based virtual screening techniques

Various inflammatory stimuli that activate the nuclear factor kappa B (NF-κB) signaling pathway converge on a serine/threonine kinase that displays a key role in the activation of NF-κB: the I kappa B kinase β (IKK-β). Therefore, IKK-β is considered an interesting target for combating inflammation and cancer. In our study, we developed a ligand-based pharmacophore model for IKK-β inhibitors. This model was employed to virtually screen commercial databases, giving a focused hit list of candidates. Subsequently, we scored by molecular shape to rank and further prioritized virtual hits by three-dimensional shape-based alignment. One out of ten acquired and biologically tested compounds showed inhibitory activity in the low micromolar range on IKK-β enzymatic activity in vitro and on NF-κB transactivation in intact cells. Compound 8 (2-(1-adamantyl)ethyl 4-[(2,5-dihydroxyphenyl)methylamino]benzoate) represents a novel chemical class of IKK-β inhibitors and shows that the presented model is a valid approach for identification and development of new IKK-β ligands.     

A Novel Role for SIRT-1 in L-Arginine Protection against STZ Induced Myocardial Fibrosis in Rats

Background

L-arginine (L-ARG) effectively protects against diabetic impediments. In addition, silent information regulator (SIRT-1) activators are emerging as a new clinical concept in treating diabetic complications. Accordingly, this study aimed at delineating a role for SIRT-1 in mediating L-ARG protection against streptozotocin (STZ) induced myocardial fibrosis.

Methods

Male Wistar rats were allocated into five groups; (i) normal control rats received 0.1 M sodium citrate buffer (pH 4.5); (ii) STZ at the dose of 60 mg/kg dissolved in 0.1 M sodium citrate buffer (pH 4.5); (iii) STZ + sirtinol (Stnl; specific inhibitor of SIRT-1; 2 mg/Kg, i.p.); (iv) STZ + L-ARG given in drinking water (2.25%) or (v) STZ + L-ARG + Stnl.

Results

L-ARG increased myocardial SIRT-1 expression as well as its protein content. The former finding was paralleled by L-ARG induced reduction in myocardial fibrotic area compared to STZ animals evidenced histopathologically. The reduction in the fibrotic area was accompanied by a decline in fibrotic markers as evident by a decrease in expression of collagen-1 along with reductions in myocardial TGF-β, fibronectin, CTGF and BNP expression together with a decrease in TGF-β and hydroxyproline contents. Moreover, L-ARG increased MMP-2 expression in addition to its protein content while decreasing expression of PAI-1. Finally, L-ARG protected against myocardial cellular death by reduction in NFκ-B mRNA as well as TNF-α level in association with decline in Casp-3 and FAS expressions andCasp-3protein content in addition to reduction of FAS positive cells. However, co-administration of L-ARG and Stnl diminished the protective effect of L-ARG against STZ induced myocardial fibrosis.

Conclusion

Collectively, these findings associate a role for SIRT-1 in L-ARG defense against diabetic cardiac fibrosis via equilibrating the balance between profibrotic and antifibrotic mediators.     

Modulatory effects of lipoic acid and selenium against cadmium-induced biochemical alterations in testicular steroidogenesis

Exposure to toxic metals including cadmium has become an increasingly recognized source of illness worldwide. Cadmium (Cd(2+) ) is one of the environmental pollutants affecting various tissues and organs including testis. The protective effect of lipoic acid and selenium on Cd(2+) -induced testicular damage was investigated. Accordingly, male Wistar rats were allocated into four groups (n = 8; each). Gp I: (control), whereas the other 3 groups received CdCl(2) (2 mg/kg, i.p. for 28 days) alone or in combination with either (i) lipoic acid (35 mg/kg, p.o) or (ii) selenium (0.35 mg/kg, p.o) throughout the experiment. Serum testosterone, luteinizing hormone and follicle-stimulating hormone levels significantly decreased in the Cd(2+) -exposed rats. The activities of testicular key androgenic enzymes, 3β-hydroxysteroid dehydrogenase and 17 β-HSD significantly decreased in Cd(2) exposed rats compared to the control counterparts. In addition, the activities of testicular marker enzymes were significantly altered in cadmium-treated animals. Significant reductions in body and testicular weight as well as antioxidant status were also observed in Cd(2+) -exposed rats. Moreover, some testicular metal levels were altered. Lipoic acid and selenium significantly increased serum testosterone level and restored testicular activity of 3β-HSD and 17 β-HSD and were effective in modulation of most of the measured biochemical parameters. The biochemical parameters were further confirmed with histopathological findings. In conclusion, the present study demonstrated the beneficial influences of lipoic acid and selenium in reducing harmful effects of Cd(2+) in rats' testes.