Ribonucleotide reductase inhibition improves the symptoms of a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200 ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200 ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.

Variations in the promoter region of the glutaminase gene and the development of hepatic encephalopathy in patients with cirrhosis: a cohort study.

BACKGROUND: Hepatic encephalopathy is a major complication of cirrhosis and is associated with a poor prognosis. OBJECTIVE: To identify mutations in the gene sequence for glutaminase in humans that could be responsible for the development of hepatic encephalopathy in patients with cirrhosis. DESIGN: Cohort study. SETTING: Outpatient clinics in 6 Spanish hospitals. PATIENTS: 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants. MEASUREMENTS: Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study. RESULTS: The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child-Turcotte-Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; P = 0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; P = 0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; P = 0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; P = 0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity. LIMITATION: Other genes and allelic variants might be involved in the clinical expression of hepatic encephalopathy. CONCLUSION: This study identifies a genetic factor that is associated with development of hepatic encephalopathy in patients with cirrhosis. PRIMARY FUNDING SOURCE: Instituto de Salud Carlos III, Spanish Ministry of Health.

Association of complement C3d receptor 2 genotypes with the acquisition of HIV infection in a trial of recombinant glycoprotein 120 vaccine.

OBJECTIVES: Complement C3d receptor 2 (CR2) is the main receptor for complement protein C3d and plays an important role in adaptive immune responses. CR2 genetic variants are associated with susceptibility to systemic lupus erythematosus as well as to HIV-1 infection. In addition, CR2 function can be subverted by HIV-1 for an efficient entry into target cells; in a process known as antibody-dependent enhancement of viral infection. We sought to determine the association between CR2 gene variants with HIV-1 acquisition after vaccination with recombinant gp120 protein (Vax004 clinical trial). DESIGN AND METHODS: This is a retrospective cross-sectional study, comprising male volunteers of European ancestry including infected (n = 273) and uninfected (n = 402) vaccinees and placebo, who were genotyped for three single nucleotide polymorphisms (SNPs) in the CR2 gene region. RESULTS: An interaction was observed between the baseline sexual behavior and the SNP rs3813946 for higher risk of infection in vacinees (interaction term P = 0.02). This SNP was associated with increased susceptibility to HIV-1 infection after vaccination in volunteers with low behavioral risk odds ratio (95% confidence interval): 5.5 (1.4-21.7) P = 0.006 but not vaccinees with high behavioral risk or volunteers given placebo (P = 0.7). Moreover, CR2 genotype was strongly associated with the rate of HIV-1 acquisition after vaccination in low-risk volunteers [hazard odds ratio (95% confidence interval): 3.3 (1.6-7.0), P = 0.001]. CONCLUSION: The current study suggests that CR2 may play a role in HIV-1 acquisition after vaccination with rgp120 proteins.

Comparative genomics of the Hedgehog loci in chordates and the origins of Shh regulatory novelties.

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications.

Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2 for biotechnological applications.

Expression vectors appear to be an indispensable tool for both biological studies and biotechnological applications. Controlling gene overexpression becomes a critical issue when protein production is desired. In addition to several aspects regarding toxicity or plasmid instability, tight control of gene expression is an essential factor in biotechnological processes. Thus, the search for better-controlled circuits is an important issue among biotechnologists. Traditionally, expression systems involve a single regulatory protein operating over a target promoter. However, these circuits are limited on their induction ratios (e.g., by their restriction in the maximal expression capacity, by their leakiness under non-induced conditions). Due to these limitations, regulatory cascades, which are far more efficient, are necessary for biotechnological applications. Thus, regulatory circuits with two modules operating in cascade offer a significant advantage. In this review, we describe the regulatory cascade based on two salicylate-responsive transcriptional regulators of Pseudomonas putida (nahR/P(sal)::xylS2), its properties, and contribution to a tighter control over heterologous gene expression in different applications.Nowadays, heterologous expression has been proven to be an indispensable tool for tackling basic biological questions, as well as for developing biotechnological applications. As the nature of the protein of interest becomes more complex, biotechnologists find that a tight control of gene expression is a key factor which conditions the success of the downstream purification process, as well as the interpretation of the results in other type of studies. Fortunately, different expression systems can be found in the market, each of them with their own pros and cons. In this review we discuss the exploitation of prokaryotic expression systems based on a promising expression system, the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2, as well as some of the improvements that have been done on this system to exploit it more efficiently in the context of both biotechnological applications and basic research.

Identification of a protective haplogenotype within CAPN10 gene influencing colorectal cancer susceptibility.

BACKGROUND AND AIM: Colorectal cancer (CRC) is one of the most prevalent types of cancer affecting both men and women in developed countries. Clinical and molecular evidence suggests that there are multiple biochemical pathways involved in its susceptibility, pathogenesis and prognosis. Several studies have reported a significant association between the incidence of CRC and type 2 diabetes mellitus (T2DM). However, genes associated with both conditions are rare. METHOD: We have analyzed the CAPN10 gene, a T2DM locus, using UCSNP-43, -44, -19, and -63 markers, looking for differences between 371 CRC patients and 605 unrelated controls of Spanish origin. RESULTS: We found that UCSNP-44 allele C is swept out from cases (OR = 0.16, P = 0.005). Moreover, the frequency of 2111/2111 haplogenotype is also statistically lower than expected in CRC patients (P = 0.006). CONCLUSION: Taken together, our results indicate a recessive model for the effect of CAPN10 variant UCSNP-44 influencing the risk of CRC and suggest a novel genetic link between T2DM and colon carcinoma.

Bone morphogenetic protein 15 (BMP15) alleles predict over-response to recombinant follicle stimulation hormone and iatrogenic ovarian hyperstimulation syndrome (OHSS).

OBJECTIVE: Controlled ovarian stimulation (COS) using recombinant follicle-stimulating hormone (rFSH) is the main treatment in assisted reproduction. We performed a pharmacogenetic analysis of bone morphogenetic protein 15 (BMP15) gene using single nucleotide polymorphisms (SNPs) in COS. We also investigated the role of the BMP15 gene in ovarian hyperstimulation syndrome (OHSS). METHODS: We analysed different intragenic SNPs located within the BMP15 gene in 307 women treated with rFSH, evaluating its involvement in COS outcome. RESULTS: First, we analysed two polymorphisms, by applying different tests for genetic association, and we found a minimum P-value in patients producing > or =12 follicles in COS (high responders) in both polymorphisms of the BMP15 gene. Using bi-directional DNA sequencing, we identified two additional single nucleotide DNA variants. Second, we conducted association studies with all polymorphisms together, and noticed that none of them seemed to fully explain the association of the BMP15 gene with over-response to rFSH. However, N103S missense mutation is predicted to disrupt the secondary structure of human BMP15 protein and is weakly associated with OHSS. This coding mutation of the BMP15 gene may partially explain the results obtained during our research. Using Thesias software, we reconstructed haplotypes with the four intragenic variants and calculated their frequencies in normal and over-responders to rFSH. The haplotype TGGA was over-represented in high responders when compared with the rest of patients. Moreover, this association was higher in patients with OHSS, with a significant global haplotypic effect of the BMP15 gene. CONCLUSION: Our results suggest a direct relationship between increased follicle production during COS and BMP15 alleles in response to rFSH in humans. The use of BMP15 markers to prevent OHSS is also a possibility that requires thorough evaluation.