Analysis of the multi-copy gene family FAM90A as a copy number variant in different ethnic backgrounds

Copy number variants contribute extensively to inter-individual genomic differences, but little is known about their inter-population variability and diversity. In a previous study (Bosch et al., 2007; 16:2572-2582), we reported that the primate-specific gene family FAM90A, which accounts for as many as 25 members in the human reference assembly, has expanded the number of FAM90A clusters across the hominoid lineage. Here we examined the copy number variability of FAM90A genes in 260 HapMap samples of European, African, and Asian ancestry, and showed significant inter-population differences (p<0.0001). Based on the recent study of Stranger et al. (2007; 315:848-853), we also explored the correlation between copy number variability and expression levels of the FAM90A gene family. Despite the high genomic variability, we found a low correlation between FAM90A copy number and expression levels, which could be due to the action of independent trans-acting factors. Our results show that FAM90A is highly variable in copy number between individuals and between populations. However, this variability has little impact on gene expression levels, thus highlighting the importance of genomic variability for genes located in regions containing segmental duplications.     

Curcumin protects cardiac cells against ischemia-reperfusion injury: effects on oxidative stress, NF-kappaB, and JNK pathways

In this study we explored the effects of curcumin in cardiac cells subjected to a protocol simulating ischemia-reperfusion (IR). Curcumin (10 microM) was administered before ischemia (pretreatment) or at the moment of reperfusion (posttreatment) and its effects were compared to those produced by a reference antioxidant (Trolox) with an equal antioxidant capacity. IR cardiac cells showed clear signs of oxidative stress, impaired mitochondrial activity, and a marked development of both necrotic and apoptotic processes; at the same time, increases in NF-kappaB nuclear translocation and JNK phosphorylation were observed. Curcumin pretreatment was revealed to be the most effective in attenuating all these modifications and, in particular, in reducing the death of IR cells. This confirms that the protective effect of curcumin is not related simply to its antioxidant properties but involves other mechanisms, notably interactions in the NF-kappaB and JNK pathways. These findings suggest that curcumin administration, in particular before the hypoxic challenge, represents a promising approach to protecting cardiac cells against IR injury. In this scenario our results point out the importance of the chronology for the outcome of the treatment and provide a differential valuation of the degree of protection that curcumin can exert by its antioxidant activity or by other mechanisms.     

Lanthanide-binding tags with unnatural amino acids: sensitizing Tb3+ and Eu3+ luminescence at longer wavelengths

Lanthanide-binding tags (LBTs) are small, genetically encoded, versatile protein fusion partners that selectively bind lanthanide ions with high affinity. The LBT motif features a strategically positioned tryptophan residue that sensitizes Tb3+ luminescence upon excitation at 280 nm. Herein, we describe the preparation of new LBT peptides that incorporate unnatural amino acids in place of tryptophan, and which sensitize both Tb3+ and Eu3+ luminescence at lower energies. We also report the semisynthesis of proteins tagged with these new LBTs using native chemical ligation. This expands the scope of LBTs and will enable their wider use in luminescence applications.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Estimating the frequency of Asian cytochrome B haplotypes in standard European and local Spanish pig breeds

Mitochondrial DNA has been widely used to perform phylogenetic studies in different animal species. In pigs, genetic variability at the cytochrome B gene and the D-loop region has been used as a tool to dissect the genetic relationships between different breeds and populations. In this work, we analysed four SNP at the cytochrome B gene to infer the Asian (A1 and A2 haplotypes) or European (E1 and E2 haplotypes) origins of several European standard and local pig breeds. We found a mixture of Asian and European haplotypes in the Canarian Black pig (E1, A1 and A2), German Piétrain (E1, A1 and A2), Belgian Piétrain (E1, A1), Large White (E1 and A1) and Landrace (E1 and A1) breeds. In contrast, the Iberian (Guadyerbas, Ervideira, Caldeira, Campanario, Puebla and Torbiscal strains) and the Majorcan Black pig breeds only displayed the E1 haplotype. Our results show that the introgression of Chinese pig breeds affected most of the major European standard breeds, which harbour Asian haplotypes at diverse frequencies (15–56%). In contrast, isolated local Spanish breeds, such as the Iberian and Majorcan Black pig, only display European cytochrome B haplotypes, a feature that evidences that they were not crossed with other Chinese or European commercial populations. These findings illustrate how geographical confinement spared several local Spanish breeds from the extensive introgression event that took place during the 18th and 19th centuries in Europe.     

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.     

Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties

Background  

The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.     

Local conservation scores without a priori assumptions on neutral substitution rates

Background  

Comparative genomics aims to detect signals of evolutionary conservation as an indicator of functional constraint. Surprisingly, results of the ENCODE project revealed that about half of the experimentally verified functional elements found in non-coding DNA were classified as unconstrained by computational predictions. Following this observation, it has been hypothesized that this may be partly explained by biased estimates on neutral evolutionary rates used by existing sequence conservation metrics. All methods we are aware of rely on a comparison with the neutral rate and conservation is estimated by measuring the deviation of a particular genomic region from this rate. Consequently, it is a reasonable assumption that inaccurate neutral rate estimates may lead to biased conservation and constraint estimates.     

Daily variation of melatonin content in the optic lobes of the crab Neohelice granulata.

Melatonin is a biogenic amine, known from almost all phyla of living organisms. In vertebrates melatonin is produced rhythmically in the pinealocytes of the pineal gland, relaying information of the environmental light/dark cycle to the organism. With regard to crustaceans only a handful of studies exist that has attempted to identify the presence and possible daily variation of this substance. We set out to investigate whether in the crab Neohelice granulata melatonin was produced in the optic lobes of these animals and underwent rhythmic fluctuations related to the daily light/dark cycle. Our experimental animals were divided into three groups exposed to different photoperiods: normal photoperiod (12L:12D), constant dark (DD), and constant light (LL). The optic lobes were collected every 4 hours over a 24-h period for melatonin quantification by radioimmunoassay (RIA). N. granulata kept under 12 L:12D and DD conditions, showed daily melatonin variations with two peaks of abundance (p<0.05), one during the day and another, more extensive one, at night. Under LL-conditions no significant daily variations were noticeable (p>0.05). These results demonstrate the presence of a daily biphasic fall and rise of melatonin in the eyestalk of N. granulata and suggest that continuous exposure to light inhibits the production of melatonin synthesis.     

Genetic susceptibility to obsessive-compulsive hoarding: the contribution of neurotrophic tyrosine kinase receptor type 3 gene

Recent work suggests that neurotrophic factors may contribute to the genetic susceptibility to obsessive-compulsive disorder (OCD). Among other clinical dimensions, the presence of hoarding obsessions and compulsions has been shown to be correlated with a number of clinical and neuroimaging findings, as well as with a different pattern of genetic inheritance. We used a linkage disequilibrium (LD)-mapping approach to investigate whether neurotrophic tyrosine kinase receptor type 3 (NTRK3), the high-affinity receptor of neurotrophin 3 (NT-3), plays a role in increasing susceptibility to hoarding in OCD. We performed an association study of 52 tag single nucleotide polymorphisms (tagSNPs) covering the whole NTRK3 gene in a sample comprising 120 OCD patients and 342 controls. Single nucleotide polymorphism association and haplotype analysis were performed. Thirty-six of our patients (30%) exhibited significant hoarding obsessions and compulsions. A significant association of two SNPs in the 3' downstream region of NTRK3 gene and obsessive-compulsive hoarding was identified: rs1017412 [odds ratio (OR) = 2.16; P = 0.001] and rs7176429 (OR = 2.78; P = 0.0001), although only the latter remained significant after Bonferroni correction. Although the haplotype analysis did not show significant results, a more extended block of LD in the OCD hoarders with respect to the control group was observed, suggesting a lower haplotype diversity in these individuals. Our findings suggest that NTRK3 may contribute to the genetic susceptibility to hoarding in OCD and may constitute an interesting gene to focus on in studies of the genetic basis of obsessive-compulsive hoarding.