Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Multicopper oxidases can act on a broad spectrum of phenolic and non‐phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), 2,6‐dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where k_cat and K_m values at 60°C were 8.1 (± 0.8) s^?1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half‐life of over 10 h at 80°C and over 7 h at 90°C. Site‐directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10‐fold increase in activity compared with the wild‐type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta‐position of aromatic substrates.     

Evolutionary History of HIV-1 Subtype B and CRF01_AE Transmission Clusters among Men Who Have Sex with Men (MSM) in Kuala Lumpur,Malaysia

HIV-1 epidemics among men who have sex with men (MSM) continue to expand in developed and developing countries. Although HIV infection in MSM is amongst the highest of the key affected populations in many countries in Southeast Asia, comprehensive molecular epidemiological study of HIV-1 among MSM remains inadequate in the region including in Malaysia. Here, we reported the phylodynamic profiles of HIV-1 genotypes circulating among MSM population in Kuala Lumpur, Malaysia. A total of n = 459 newly-diagnosed treatment-naïve consenting subjects were recruited between March 2006 and August 2012, of whom 87 (18.9%) were self-reported MSM. Transmitted drug resistance mutations were absent in these isolates. Cumulatively, phylogenetic reconstructions of the pro-rt gene (HXB2∶2253–3275) showed that HIV-1 subtype B and CRF01_AE were predominant and contributed to approximately 80% of the total HIV-1 infection among MSM. In addition to numerous unique transmission lineages within these genotypes, twelve monophyletic transmission clusters of different sizes (2–7 MSM sequences, supported by posterior probability value of 1) were identified in Malaysia. Bayesian coalescent analysis estimated that the divergence times for these clusters were mainly dated between 1995 and 2005 with four major transmission clusters radiating at least 12 years ago suggesting that active spread of multiple sub-epidemic clusters occurred during this period. The changes in effective population size of subtype B showed an exponential growth within 5 years between 1988 and 1993, while CRF01_AE lineage exhibited similar expansion between 1993 and 2003. Our study provides the first insight of the phylodynamic profile of HIV-1 subtype B and CRF01_AE circulating among MSM population in Kuala Lumpur, Malaysia, unravelling the importance of understanding transmission behaviours as well as evolutionary history of HIV-1 in assessing the risk of outbreak or epidemic expansion.     

Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives

The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC).

Materials and Methods

The OncoCarta^™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.

Results

Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.

Conclusion

Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.     

A Modified Entropy-Based Approach for Identifying Gene-Gene Interactions in Case-Control Study

Gene-gene interactions may play an important role in the genetics of a complex disease. Detection and characterization of gene-gene interactions is a challenging issue that has stimulated the development of various statistical methods to address it. In this study, we introduce a method to measure gene interactions using entropy-based statistics from a contingency table of trait and genotype combinations. We also developed an exploration procedure by using graphs. We propose a standardized relative information gain (RIG) measure to evaluate the interactions between single nucleotide polymorphism (SNP) combinations. To identify the k ^th order interactions, contingency tables of trait and genotype combinations of k SNPs are constructed, with which RIGs are calculated. The RIGs are standardized using the mean and standard deviation from the permuted datasets. SNP combinations yielding high standardized RIG are chosen for gene-gene interactions. Detection of high-order interactions and comparison of interaction strengths between different orders are made possible by using standardized RIG. We have applied the proposed standardized entropy-based method to two types of data sets from a simulation study and a real genetic association study. We have compared our method and the multifactor dimensionality reduction (MDR) method through power analysis of eight different genetic models with varying penetrance rates, number of SNPs, and sample sizes. Our method shows successful identification of genetic associations and gene-gene interactions both in simulation and real genetic data. Simulation results suggest that the proposed entropy-based method is better able to detect high-order interactions and is superior to the MDR method in most cases. The proposed method is well suited for detecting interactions without main effects as well as for models including main effects.     

The Relationship between Age at First Birth and Mother's Lifetime Earnings: Evidence from Danish Data

Background

Having children creates career interruptions and reductions in labor income for women. This study documents the relation between the age at first birth (AFB) and women’s labor income. We study these dynamics in the short run (i.e. ratio between labor income at AFB and two years prior to AFB) and long run (i.e., positive/negative differences in total lifetime labor income).

Methods

Using unique Danish administrative register data for the entire Danish population, we estimate the age-income profiles separately for college and non-college women conditional on marital status, and mothers’ age at first birth (AFB). We compute the lifetime labor income differentials by taking the differences between the labor income of women with and without children at each AFB.

Results

The short-run loss in labor income, defined as the difference in percentages between the income earned two years prior to AFB and income earned at AFB, ranges from 37% to 65% for college women and from 40% to 53% for non-college women. These losses decrease monotonically with respect to AFB for both education groups. Our results on the lifetime labor income differentials between mothers and women without children also show a net effect that is monotonic (from negative to positive) in AFB. With AFB<25, the lifetime labor income loss for college women is -204% of their average annual labor income and this figure is -252% for non-college women. There are lifetime labor income gains with AFB>31. The largest gains for college women are 13% of their average annual income and this figure is 50% for non-college women.

Conclusion

Women have a large and unambiguous short-run reduction in labor income at their AFB. In terms of lifetime labor income, both college and non-college women, compared to childless women, are associated with lower income of more than twice their respective average annual income when bearing a child at AFB<25. In other words, women with AFB<25 are associated with a lower lifetime income of more than two years of annual labor income. The lifetime labor incomes for college and non-college women associated with AFB>31 are relatively higher.     

Paternal B Vitamin Intake Is a Determinant of Growth,Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

Background

The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well.

Objective

In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content.

Methods

Male Apc^1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B₂, B₆, B₁₂, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc^1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden.

Results

No differences in intestinal tumor incidence or burden were found between male Apc^1638N offspring of different paternal diet groups. Although in female Apc^1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring.

Conclusions

In this animal model, modulation of paternal B vitamin intake prior to mating alters offspring weight gain, lipid metabolism and tumor growth in a sex-specific fashion. These results highlight the need to better define how paternal nutrition affects the health of offspring.