Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients

Altres ajuts: Fundación para la Investigación y Prevención del SIDA en España (FIPSE 36610, 36572/06); Red de Investigación en SIDA (RIS RD12/0017/0005, RD12/0017/0014).To assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS). Three-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method. HALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53-3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/10 6 cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): 'A+A'; OR for 'A+B' vs. ref.: 1.39 [0.69-2.80]; OR for 'B+A' vs. ref.: 2.16 [1.22-3.83]; OR for 'B+B' vs. ref.: 3.13, 95%CI: 1.54-6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56-4.14, P = 0.001), (c) use of EFV (1.10 [1.00-1.21], P = 0.008, per year of use). HALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine

Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

In-depth clinical and biological exploration of DNA Damage Immune Response (DDIR) as a biomarker for oxaliplatin use in colorectal cancer

PURPOSE: The DNA Damage Immune Response (DDIR) assay was developed in breast cancer (BC) based on biology associated with deficiencies in homologous recombination and Fanconi Anemia (HR/FA) pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and oesophageal cancers. In colorectal cancer (CRC) there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in CRC and characterised the biology in DDIR-positive CRC. METHODS: Samples and clinical data were assessed according to DDIR status from patients who received either 5FU or FOLFOX within the FOCUS trial (n=361, stage 4), or neo-adjuvant FOLFOX in the FOxTROT trial (n=97, stage 2/3). Whole transcriptome, mutation and immunohistochemistry data of these samples were used to interrogate the biology of DDIR in CRC. RESULTS: Contrary to our hypothesis, DDIR negative patients displayed a trend towards improved outcome for oxaliplatin-based chemotherapy compared to DDIR positive patients. DDIR positivity was associated with Microsatellite Instability (MSI) and Colorectal Molecular Subtype 1 (CMS1). Refinement of the DDIR signature, based on overlapping interferon-related chemokine signalling associated with DDIR positivity across CRC and BC cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in CRC. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in CRC. However, data presented here suggests the potential of the DDIR assay in identifying immune-rich tumours that may benefit from immune checkpoint blockade, beyond current use of MSI status

Discovery of VHE Gamma Radiation from IC443 with the MAGIC Telescope

We report the detection of a new source of very high energy (VHE, E_gamma >= 100GeV) gamma-ray emission located close to the Galactic Plane, MAGIC J0616+225, which is spatially coincident with SNR IC443. The observations were carried out with the MAGIC telescope in the periods December 2005 - January 2006 and December 2006 - January 2007. Here we present results from this source, leading to a VHE gamma-ray signal with a statistical significance of 5.7 sigma in the 2006/7 data and a measured differential gamma-ray flux consistent with a power law, described as dN_gamma/(dA dt dE) = (1.0 +/- 0.2)*10^(-11)(E/0.4 TeV)^(-3.1 +/- 0.3) cm^(-2)s^(-1)TeV^(-1). We briefly discuss the observational technique used and the procedure implemented for the data analysis. The results are put in the perspective of the multiwavelength emission and the molecular environment found in the region of IC443.Comment: Accepted by ApJ Letter

The influence of religious identity and socio-economic status on diet over time, an example from medieval France

In Southern France as in other parts of Europe, significant changes occurred in settlement patterns between the end of Antiquity and the beginning of the Middle Ages. Small communities gathered to form, by the tenth century, villages organized around a church. This development was the result of a new social and agrarian organization. Its impact on lifestyles and, more precisely, on diet is still poorly understood. The analysis of carbon and nitrogen isotopes in bone collagen from the inhabitants of the well-preserved medieval rural site Missignac-Saint Gilles le Vieux (fifth to thirteenth centuries, Gard, France) provides insight into their dietary practices and enables a discussion about its transformation over time. A sample of 152 adult individuals dated from 675 to 1175 AD (75 females, 77 males) and 75 specimens from 16 non-human species were analyzed. Results show the exploitation of freshwater, marine, and terrestrial ecosystems as well as various breeding practices specific to each species. The use of both C4 and halophyte plants for feeding domestic animals was also observed. Concerning human dietary practices, a change seemed to occur at the beginning of the tenth century with an increase of δ15N values and a decrease of δ13C values. This corresponds to the introduction of a significant amount of freshwater resources into the diet and could be related to the evolution of the Catholic doctrine. A concomitant diversification of access to individual food resources was also observed, probably linked to the increased diversity of practice inside a population otherwise perceived as one community

Ongoing Phenotypic and Genomic Changes in Experimental Coevolution of RNA Bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite–host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163–165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria

Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis

<p>Abstract</p> <p>Background</p> <p>Osteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of <it>Emblica officinalis </it>fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts.</p> <p>Methods</p> <p>The effects of extracts from <it>Emblica officinalis </it>on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of <it>Emblica officinalis </it>extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, <it>in vitro </it>effects of <it>Emblica officinalis </it>extracts on NF-kB transcription factor activity were determined by gel shift experiments.</p> <p>Results</p> <p>Extracts of <it>Emblica officinalis </it>were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. <it>Emblica officinalis </it>increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that <it>Emblica officinalis </it>extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that <it>Emblica officinalis </it>extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of <it>Emblica officinalis </it>on the expression levels of interleukin-6, a NF-kB specific target gene.</p> <p>Conclusion</p> <p>Induction of apoptosis of osteoclasts could be an important strategy both in interfering with rheumatoid arthritis complications of the bone skeleton leading to joint destruction, and preventing and reducing osteoporosis. Accordingly, we suggest the application of <it>Emblica officinalis </it>extracts as an alternative tool for therapy applied to bone diseases.</p