Plant extracts with anti-inflammatory properties—A new approach for characterization of their bioactive compounds and establishment of structure–antioxidant activity relationships

Geranium robertianum L. (Geraniacea) and Uncaria tomentosa (Willd.) DC. (Rubiaceae) plant extracts, frequently used in traditional medicine for treatment of inflammatory and cancer diseases, were studied to identify potential bioactive compounds that may justify their therapeutic use and their underlying mechanisms of action. Since some of the pharmacological properties of these plant extracts may be linked to their antioxidant potential, the antioxidant activity, in relation to free radical scavenging, was measured by the ABTS/HRP and DPPH assays, presenting U. tomentosa the higher activity. The antioxidant activity was also evaluated by scavenging of HOCl, the major strong oxidant produced by neutrophils and a potent pro-inflammatory agent. U. tomentosa was found to be a better protector against HOCl, which may justify its effectiveness against inflammatory diseases. SPE/LC-DAD was used for separation/purification purposes and ESI-MS/MS for identification/characterization of the major non-volatile components, mainly flavonoids and phenolic acids. The ESI-MS/MS methodology proposed can be used as a model procedure for identification/characterization of unknowns without the prerequisite for standard compounds analysis. The ESI-MS/MS data obtained were consistent with the antioxidant activity results and structure–activity relationships for the compounds identified were discussed.     

Intragenomic 16S rDNA Divergence in <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> Is an Adaptation to Different Temperatures

The halophilic archaeon Haloarcula marismortui contains three ribosomal RNA operons, designated rrnA, rrnB, and rrnC. Operons A and C are virtually identical, whereas operon B presents a high divergence in nucleotide sequence, having up to 135 nucleotide polymorphisms among the three 16S, 23S, and 5S ribosomal RNA genes. Quantitative PCR and structural analyses have been performed to elucidate whether the presence of this intragenomic heterogeneity could be an adaptation to the variable environmental conditions in the natural habitat of H. marismortui. Variation in salt concentration did not affect expression but variation in incubation temperature did produce significant changes, with operon B displaying an expression level four times higher than the other two together at 50°C and three times lower at 15°C. We show that the putative promoter region of operon B is also different. In addition, the predicted secondary structure of these genes indicated that they have distinct stabilities at different temperatures and a mutant strain lacking operon B grew slower at high temperatures. This study supports the idea that divergent rRNA genes can be adaptive, with different variants being functional under different environmental conditions (e.g., temperature). The same phenomenon could take place in other halophiles or thermophiles with intragenomic rDNA heterogeneity, where the use of 16S rDNA as a phylogenetic marker and indicator of biodiversity should be used with caution.     

Angiotensin Converting Enzyme Inhibitors as Oxygen Free Radical Scavengers

The authors have compared the ability of two non-SH-containing angiotensin converting enzyme (ACE) inhibitors (enalaprilat and lisinopril) with an -SH containing ACE inhibitor (captopril) to scavenge the hydroxyl radical (OH). All three compounds were able to scavenge -OH radicals generated in free solution at approximately diffusion-controled rates (10¹⁰ M^-1s^-1) as established by the deoxyribose assay in the presence of EDTA. The compounds also inhibited deoxyribose degradation in reaction mixtures which did not contain EDTA but not so effectively. This later finding also suggests that they have some degree of metal-binding capability. Chemiluminescence assays of oxidation of hypoxanthine by xanthine oxidase in the presence of luminol, confirm that the three ACE inhibitors are oxygen free radical scavengers. Our results indicate that the presence of a sulphydryl group in the chemical structure of ACE inhibitors is not relevant for their oxygen free radical scavenging ability.     

Col10a1 gene expression and chondrocyte hypertrophy during skeletal development and disease

The type X collagen gene, COLIOA1, is specifically expressed by hypertrophic chondrocytes during endochondral ossification. Endochondral ossification is a well-coordinated process that involves a cartilage intermediate and leads to formation of most of the skeleton in vertebrates during skeletogenesis. Chondrocyte hypertrophy is a critical stage of endochondral ossification linking both bone and cartilage development. Given its specific association with chondrocyte hypertrophy, type X collagen plays essential roles in endochondral ossification. It was previously shown that transgenic mice with mutant type X collagen develop variable skeleton-hematopoietic abnormalities indicating defective endochondral ossification, while mutations and abnormal expression of human COLIOA1 cause abnormal chondrocyte hypertrophy that has been seen in many skeletal disorders, including skeletal chondrodysplasia and osteoarthritis. In this review, we summarized the skeletal chondrodysplasia with COLIOA1 gene mutation that shows growth plate defect. We also reviewed recent studies that correlate the type X collagen gene expression and chondrocyte hypertrophy with osteoarthritis. Due to its significant clinical relevance, the type X collagen gene regulation has been extensively studied over the past two decades. Here, we focus on recent progress characterizing the cis-enhancer elements and their binding factors that together confer hypertrophic chondroeyte-specific murine type X collagen gene （CollOal） expression. Based on literature review and our own studies, we surmise that there are multiple factors that contribute to hypertrophic chondrocyte-specific CoHOal expression. These factors include both transactivators （such as Runx2, MEF2C etc.） and repressors （such as AP1, NFATcl, Sox9 etc.）, while other co-factors or epigenetic control of CollOal expression may not be excluded.     

Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?

RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits are proposed for FFPE samples yet no comparison on low quantities were performed. To select the most reliable, cost-effective, and relevant RNA-Seq approach, we applied five FFPE-compatible kits (based on 3′ capture, exome-capture and ribodepletion approaches) using 8 ng to 400 ng of FFPE-derived RNA and compared them to Nanostring on FFPE samples and to a reference PolyA (Truseq) approach on flash-frozen samples of the same tumors. We compared gene expression correlations and reproducibility. The Smarter Pico V3 ribodepletion approach appeared systematically the most comparable to Nanostring and Truseq (p < 0.001) and was a highly reproducible technique. In comparison with exome-capture and 3′ kits, the Smarter appeared more comparable to Truseq (p < 0.001). Overall, our results suggest that the Smarter is the most robust RNA-Seq technique to study small FFPE samples and 3′ Lexogen presents an interesting quality–price ratio for samples with less limiting quantities.     

Mining virulence genes using metagenomics

When a bacterial genome is compared to the metagenome of an environment it inhabits, most genes recruit at high sequence identity. In free-living bacteria (for instance marine bacteria compared against the ocean metagenome) certain genomic regions are totally absent in recruitment plots, representing therefore genes unique to individual bacterial isolates. We show that these Metagenomic Islands (MIs) are also visible in bacteria living in human hosts when their genomes are compared to sequences from the human microbiome, despite the compartmentalized structure of human-related environments such as the gut. From an applied point of view, MIs of human pathogens (e.g. those identified in enterohaemorragic Escherichia coli against the gut metagenome or in pathogenic Neisseria meningitidis against the oral metagenome) include virulence genes that appear to be absent in related strains or species present in the microbiome of healthy individuals. We propose that this strategy (i.e. recruitment analysis of pathogenic bacteria against the metagenome of healthy subjects) can be used to detect pathogenicity regions in species where the genes involved in virulence are poorly characterized. Using this approach, we detect well-known pathogenicity islands and identify new potential virulence genes in several human pathogens.     

Candida albicans cell wall ssa proteins bind and facilitate import of salivary histatin 5 required for toxicity

Fungicidal activity of Hst 5 is initiated by binding to cell surface proteins on Candida albicans, followed by intracellular transport to cytoplasmic effectors leading to cell death. As we identified heat shock 70 proteins (Ssa1p and/or Ssa2p) from C. albicans lysates that bind Hst 5, direct interactions between purified recombinant Ssa proteins and Hst 5 were tested by pull-down and yeast two-hybrid assays. Pulldown of both native complexes and those stabilized by cross-linking demonstrated higher affinity of Hst 5 for Ssa2p than for Ssa1p, in agreement with higher levels of interactions between Ssa2p and Hst 5 measured by yeast two-hybrid analyses. C. albicans ssa1Delta and ssa2Delta mutants were constructed to examine Hst 5 binding, translocation, and candidacidal activities. Both ssa1Delta and ssa2Delta mutants were indistinguishable from wild-type cells in growth and hyphal formation. However, C. albicans ssa2Delta mutants were highly resistant to the candidacidal activity of Hst 5, although the ssa1Delta mutant did not have any significant reduction in killing by Hst 5. Total cellular binding of 125I-Hst 5 in the ssa2Delta mutant was reduced to one-third that of wild-type cells, in contrast to the ssa1Delta mutant whose total cellular binding of Hst 5 was similar to the wild-type strain. Intracellular transport of Hst 5 was significantly impaired in the ssa2Delta mutant strain, but only mildly so in the ssa1Delta mutant. Thus, C. albicans Ssa2p facilitates fungicidal activity of Hst 5 in binding and intracellular translocation, whereas Ssa1p appears to have a lesser functional role in Hst 5 toxicity.