4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4ß-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4ß-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4ß-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4ß-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4ß-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4ß-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4ß-dPE A. Furthermore, NF-κb transcription factor is involved in 4ß-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4ß-dPE A in combination with other LRAs. When 4ß-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4ß-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4ß-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

A multinested PCR for detection of the equine piroplasmids Babesia caballi and Theileria equi.

Two haemoparasites, Theileria equi and Babesia caballi, cause equine piroplasmosis (EP), one of the most prevalent tick-borne diseases in horses. The main aim of the present study was to develop and evaluate a multinested PCR (mn-PCR) for simultaneous detection of the equine piroplasmids T. equi and B. caballi, by amplification of five genetic markers (18S rRNA, ß-tubulin, cytB, EMA-1 and RAP-1). This novel assay detected a high prevalence of equine piroplasmids in 235 horse blood samples collected in Castilla-León and Extremadura, Spain. The overall prevalence of infection with equine piroplasmids by mn-PCR was 72.8% (171/235), with 66.0% (155/235) of the animals positive for T. equi and 29.4% (69/235) positive for B. caballi. The seroprevalence obtained by cELISA for the same set of samples was lower than the infection prevalence recorded by mn-PCR, for either of the two equine piroplasmids (62.6%) as well as for T. equi alone (61.7%) or B. caballi alone (3.8%). There was high agreement among the mn-PCR and cELISA assays for diagnosis of EP caused by T. equi (κ = 0.83) but not for B. caballi (κ = 0.06). A phylogenetic analysis based on the RAP-1 gene of B. caballi showed that the strains from Spain clustered with those from Israel.

Incident hand OA is strongly associated with reduced peripheral blood leukocyte telomere length.

OBJECTIVE: To evaluate the relationship of telomere length to the prevalence and incidence of hand osteoarthritis in a longitudinal cohort. DESIGN: We conducted a cross-sectional and longitudinal analysis of data from a subset of participants in the Osteoarthritis Initiative (OAI) recruited between February 2004 and May 2006. 274 individuals were eligible for the study based on availability of both baseline and 48-month hand radiographs and peripheral blood leucocyte telomere length data. Mean telomere length of peripheral blood leukocytes (PBL)s from the DNA samples was determined using a validated quantitative polymerase chain reaction (PCR)-based assay, and hand radiographs were analyzed and graded using the Kellgren-Lawrence scale. RESULTS: In joint -level analyses, prevalent Interphalangeal Joint Osteoarthritis (IPJOA) was significantly associated with PBL telomere length in the baseline sample in unadjusted analyses (RR = 2.84; 95% CI:0.87-9.29) or in models adjusted for age, sex, and body mass index (aRR = 1.10; 95% CI: 0.96-1.27). The association in crude and adjusted analyses appeared slightly stronger with incident IPJOA, especially in the subset with normal hands at baseline (aRR = 1.62; 95% CI: 1.02-2.57). PBL telomere length was also associated with prevalent HOA at baseline (significant in unadjusted analysis: RR = 1.22; 95% CI 1.06-1.42), but not after adjusting for covariates: aRR = 1.12; 95% CI: 0.96-1.30). The magnitude of association was stronger for incident HOA, especially incident symptomatic HOA (aRR = 1.53; 95% CI: 1.09-2.15). CONCLUSIONS: In summary, the results of this exploratory analysis are confirmatory of previous work showing a cross-sectional relationship between telomere length and HOA and add to the field by demonstrating an even stronger association with incident IPJOA, both radiographic and symptomatic.

Parents' genetic dissimilarity and offspring sex in a polygynous mammal.

Offspring quality may benefit from genetic dissimilarity between parents. However, genetic dissimilarity may trade-off with additive genetic benefits. We hypothesized that when sexual selection produces sex-specific selective scenarios, the relative benefits of additive genetic vs. dissimilarity may differ for sons and daughters. Here we study a sample of 666 red deer (Cervus elaphus) microsatellite genotypes, including males, females and their foetuses, from 20 wild populations in Spain (the main analyses are based on 241 different foetuses and 190 mother-foetus pairs). We found that parental lineages were more dissimilar in daughters than in sons. On average, every mother was less related to her mate than to the sample of fathers in the population when producing daughters not sons. Male foetuses conceived early in the rutting season were much more inbred than any other foetuses. These differences maintained through gestation length, ruling out intrauterine mortality as a cause for the results, and indicating that the potential mechanism producing the association between parents' dissimilarity and offspring sex should operate close to mating or conception time. Our findings highlight the relevance of considering the sex of offspring when studying genetic similarity between parents.

The karyotype of the Iberian imperial eagle (Aquila adalberti) analyzed by classical and DNA replication banding.

We report here for the first time the karyotype of the Iberian imperial eagle (Aquila adalberti). All eagles examined had a diploid number of 82 chromosomes and a greater number of microchromosomes (12 pairs) than has been found in all other species of the Accipitridae family. This karyotypic evidence corroborates the recent separation of A. adalberti from A. heliaca on the basis of molecular data. RB-FPG banding induced a specific banding pattern that allowed us to identify homologous chromosome pairs and revealed features about late and early replicating regions. Several chromosome banding techniques (C-, CMA3-, and restriction endonuclease banding and silver staining) were used to characterize the karyotype more accurately. Two GC-rich, late-replicating heterochromatin regions were found in the W chromosome. These regions are AluI resistant and can be used for sex determination in this species. All microchromosomes were heterochromatic, GC rich, and late replicating. Silver staining revealed active nucleolus organizing regions on a pair of microchromosomes that were entirely heterochromatic and stained intensely after CMA3-banding. Different chromosome rearrangements are discussed in order to establish the phylogenetic relationship between A. adalberti and its most closely related species, A. heliaca.

Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts.

The mucopolysaccharide capsule of Cryptococcus neoformans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time-saving technique to obtain high yields of good-quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months.

Characterization of the heterochromatic chromosome regions in sheep.

In order to elucidate the structural chromosome organization of the heterochromatic regions in sheep, we have used C-banding, silver-staining, sequential CDD technique and restriction endonuclease banding. By these banding techniques we obtained four fractions of repetitive DNA, the autosomal fractions A and B, the C fraction in the X chromosome, and the D fraction in the Y chromosome. Silver staining revealed active nucleolus organizer regions (NOR's) on the telomeric GC-rich areas of chromosomes 1, 2, 3, 4, and 25 which were digested with HaeIII restriction endonuclease.

Characterization of the karyotype of the tench (Tinca tinca L.) and analysis of its chromosomal heterochromatic regions by C-banding, Ag-staining, and restriction endonuclease banding.

Beginning with a description of the conventional Giemsa-stained karyotype of the tench (Tinca tinca L.), the structure and variability of the chromosomal heterochromatic regions in this cyprinid species were analyzed by means of C-, silver-, and restriction endonuclease banding. Silver staining revealed active nucleolus organizer regions (NORs) on the secondary constriction of chromosome pair 3. Constitutive heterochromatin was associated with NOR regions detected by C-banding. Restriction endonuclease digestion with AluI, TaqI, and HaeIII induced specific banding patterns that allowed identification of homologous chromosome pairs and revealed features about the sequence composition of several chromosomal heterochromatic regions and of the NOR-associated heterochromatin.