Chemical Investigation of Marine-Derived Fungus Aspergillus flavipes for Potential Anti-Inflammatory Agents

The marine fungus, Aspergillus flavipes (MTCC 5220), was isolated from the pneumatophore of a mangrove plant Acanthus ilicifolius found in Goa, India. The crude extract of A. flavipes was found to show anti-inflammatory activity. It blocked interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-activated THP-1 cells with IC₅₀ of 2.69±0.5 μM and 6.64±0.4 μM, respectively. The chemical investigation led to the isolation of optically inactive 4β-[(1E)-propen-1-yl]cyclopentane-1β,2β-diol ( 1 ) along with a new optically active diastereoisomeric compound, 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol ( 2 ). In addition, the fungus also produced known compounds (+)-terrein ( 3 ), butyrolactone I ( 4 ) and butyrolactone II ( 5 ) in high yields. Among these, (+)-terrein ( 3 ) exhibited IL-6 and TNF-α inhibition activity with IC₅₀ of 8.5±0.68 μM and 15.76±0.18 μM, respectively, while butyrolactone I ( 4 ) exhibited IC₅₀ of 12.03±0.85 μM (IL-6) and 43.29±0.76 μM (TNF-α) inhibition activity with low toxicity to host cells in LPS stimulated THP-1 cells. This is the first report of the isolation and characterization of 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol ( 2 ). The structures of all the isolated compounds were elucidated on the basis of extensive detailed NMR spectroscopic data. Anti-inflammatory activity of the fungi A. flavipes is presented here for the first time, which was due to (+)-terrein and butyrolactone I, as the major constituents and they can be further explored in the therapeutic area.     

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.     

Quality of Artemisinin-Based Combination Formulations for Malaria Treatment: Prevalence and Risk Factors for Poor Quality Medicines in Public Facilities and Private Sector Drug Outlets in Enugu,Nigeria

BackgroundArtemisinin-based combination therapies are recommended by the World Health Organisation (WHO) as first-line treatment for Plasmodium falciparum malaria, yet medication must be of good quality for efficacious treatment. A recent meta-analysis reported 35% (796/2,296) of antimalarial drug samples from 21 Sub-Saharan African countries, purchased from outlets predominantly using convenience sampling, failed chemical content analysis. We used three sampling strategies to purchase artemisinin-containing antimalarials (ACAs) in Enugu metropolis, Nigeria, and compared the resulting quality estimates.MethodsACAs were purchased using three sampling approaches - convenience, mystery clients and overt, within a defined area and sampling frame in Enugu metropolis. The active pharmaceutical ingredients were assessed using high-performance liquid chromatography and confirmed by mass spectrometry at three independent laboratories. Results were expressed as percentage of APIs stated on the packaging and used to categorise each sample as acceptable quality, substandard, degraded, or falsified.ResultsContent analysis of 3024 samples purchased from 421 outlets using convenience (n=200), mystery (n=1,919) and overt (n=905) approaches, showed overall 90.8% ACAs to be of acceptable quality, 6.8% substandard, 1.3% degraded and 1.2% falsified. Convenience sampling yielded a significantly higher prevalence of poor quality ACAs, but was not evident by the mystery and overt sampling strategies both of which yielded results that were comparable between each other. Artesunate (n=135; 4 falsified) and dihydroartemisinin (n=14) monotherapy tablets, not recommended by WHO, were also identified.ConclusionRandomised sampling identified fewer falsified ACAs than previously reported by convenience approaches. Our findings emphasise the need for specific consideration to be given to sampling frame and sampling approach if representative information on drug quality is to be obtained.     

Sumoylation Influences DNA Break Repair Partly by Increasing the Solubility of a Conserved End Resection Protein

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.     

Tumor Necrosis Factor Inhibition and Head and Neck Cancer Recurrence and Death in Rheumatoid Arthritis

The objective of this retrospective cohort study was to determine the effect of tumor necrosis factor inhibitor (TNFi) therapy on the risk of head and neck cancer (HNC) recurrence or HNC-attributable death in patients with rheumatoid arthritis (RA). RA patients with HNC were assembled from the US national Veterans’ Affairs (VA) administrative databases, and diagnoses confirmed and data collected by electronic medical record review. The cohort was divided into those treated with non-biologic disease-modifying anti-rheumatic drugs (nbDMARDs) versus TNF inhibitors (TNFi) after a diagnosis of HNC. Likelihood of a composite endpoint of recurrence or HNC-attributable death was determined by Cox proportional hazards regression. Of 180 patients with RA and HNC, 31 were treated with TNFi and 149 with nbDMARDs after the diagnosis of HNC. Recurrence or HNC-attributable death occurred in 5/31 (16.1%) patients in the TNFi group and 44/149 (29.5%) patients in the nbDMARD group (p = 0.17); it occurred in 2/16 (13%) patients who received TNFi in the year prior to HNC diagnosis but not after. Overall stage at diagnosis (p = 0.03) and stage 4 HNC (HR 2.49 [CI 1.06–5.89]; p = 0.04) were risk factors for recurrence or HNC-attributable death; treatment with radiation or surgery was associated with a lower risk (HR 0.35 [CI 0.17–0.74]; p = 0.01 and HR 0.39 [CI 0.20–0.76]; p = 0.01 respectively). Treatment with TNFi was not a risk factor for recurrence or HNC-attributable death (HR 0.75; CI 0.31–1.85; p = 0.54). We conclude that treatment with TNFi may be safe in patients with RA and HNC, especially as the time interval between HNC treatment and non-recurrence increases. In this study, TNF inhibition was not associated with an increase in recurrence or HNC-attributable death.     

Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5′s helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

Arbuscular mycorrhizal and septate endophyte fungal associations in lycophytes and ferns of south India

We studied extent and type of arbuscular mycorrhizal (AM) and septate endophytic (SE) fungal associations in five lycophytes and 50 ferns collected from Eastern and Western Ghats regions. Of the 54 species and one variety (belonging to 31 genera) examined; 54 taxa had AM association and AM fungal structures were absent in Marsilea quadrifolia. This is the first report of AM and SE fungal status for 26 species each. Of the 55 taxa examined, AM morphology has been evaluated for the first time in 51 species. The hydrophytic fern Salvinia molesta was mycorrhizal and non-mycorrhizal at different sites. All the epiphytic and saxicolous species examined were mycorrhizal. The percentage of AM colonization ranged from 22.23 (Christella parasitica) to 82.20 (Adiantum lunulatum) in ferns and 53.46 (Selaginella bryopteris) to 84.34 (Selaginella sp.) in lycophytes. Epiphytic life-forms had the maximum average AM colonization levels, whereas aquatic life-forms had the minimum colonization levels. The percentage root length colonized by septate fungi ranged between 0.59 in Ophioglossum reticulatum and 16.36 in Pteris pellucida. The root length with AM and SE fungal structures as well as their total colonization significantly varied among the taxa examined. Most of the lycophytes and ferns had intermediate-type of AM morphology with a few exhibiting Paris-type. AM fungal spore numbers ranged from 1.0 (Angiopteris evecta, Pteridium aquilinum) to (Nephrolepis exaltata) 9.3 spores per 25 g soil and varied significantly among taxa. AM fungal spore morphotypes belonging to Claroideoglomus, Funneliformis, Glomus and Rhizophagus were recorded.