Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts

Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A2 (A2a and A2b) receptor (A2aR and A2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A2a and/or A2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A2aR or A2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A1R, A2bR, A2a), and A3R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A2R and A2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A2aR or A2bR. Overexpression of A2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A2bR yielded increases in protein and collagen synthesis. In contrast, A2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A2bR are critically involved in ADO-mediated inhibition of CF functions.     

Porous polyethylene implants in orbital floor reconstruction

The purpose of this article is to present the authors' experience with the use of porous polyethylene ultrathin sheets for orbital floor reconstruction. Thirty-two patients with orbital floor fractures were treated with porous polyethylene ultrathin sheets. Sixteen cases corresponded to orbitozygomatic fractures, 11 cases corresponded to pure orbital floor fractures, and five corresponded to panfacial fractures. The subciliary approach was used in 15 patients and the transconjunctival approach in nine; another three patients were operated on through a preexisting eyebrow wound, two were operated on with a subtarsal approach, two were operated on through an eyebrow extension of a facial wound, and one patient was operated on through the facial wound. Intraoperatively, all patients received a prophylactic dose of intravenous antibiotics. Postoperatively, 24 patients received amoxicillin clavulanate for 5 to 7 days, two patients received clindamycin, and six patients received no antibiotics. Enophthalmos was corrected in 15 of 24 patients (62.5 percent), and hypoglobus in nine of 11 (82 percent). Diplopia was resolved in 25 of 28 patients (89.3 percent) with preoperative impairment. Extrinsic eye movement impairment was resolved in 25 of 27 patients (92.6 percent). A preoperative visual acuity deficit was present in four patients (12.5 percent) and was resolved in one (from 20/100 to 20/20). Visual acuity improved in one patient (from 20/60 to 20/30). In the other two patients, visual acuity remained altered (from 20/30 to 20/30). One patient (3.1 percent) suffered blindness induced by surgery. Nine of 26 patients (34.6 percent) had residual infraorbital nerve hypesthesia and five (19.2 percent) had residual paresthesias. Postoperatively, epiphora was present in six patients (18.8 percent) and ectropion in five (15.6 percent). Although there was no statistical significance between the surgical approach and the presence of epiphora (p = 0.211) and ectropion (p = 0.422), patients who were treated using the transconjunctival approach suffered reduced ectropion (0 percent) compared with patients treated using the subciliary approach (20 percent). However, patients treated using the transconjunctival approach suffered increased epiphora (22.2 percent) compared with those treated with the subciliary approach (13.3 percent). There were four cases (12.5 percent) of postoperative facial infections. Two of these cases were resolved with systemic antibiotics, one was resolved with bone sequestrum resection, and one patient needed removal of the implant. Orbital infections were related in all cases to titanium osteosynthesis miniplates or skull bone graft. When comparing patients who were treated with and without antibiotics, no statistical differences (p = 0.958) were found relative to the presence of infections. Correction of hypoglobus is technically easier than enophthalmos, because enophthalmic correction requires a wide, deep subperiosteal dissection and implant positioning, posterior to the equator of the globe, with the inherent risk of orbital apex injury.     

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.     

Length of postpartum anestrus in goats in subtropical Mexico: effect of season of parturition and duration of nursing

The aim of this study was to determine whether season of birth and length of nursing affected the duration of postpartum anestrus in Creole female goats maintained on a constant plane of nutrition in subtropical Mexico. Three experiments were conducted in the Laguna region in the State of Coahuila, Mexico (26 degrees N). In the first experiment, 34 goats gave birth in January; in the second, 31 females gave birth in May; and in the third, 22 goats kidded in October. At parturition, females were allocated to 1 of 3 groups based on body weight and date of parturition: kids were weaned at 2, 30 or 90 d according to their group. After weaning, females were milked manually once a day until the end of the study. All animals were kept in a shed and were fed alfalfa ad libitum and given 200 g of concentrate daily. Starting 1 wk after parturition, estrous behavior was detected twice daily using an apron-bearing male, and blood samples were obtained twice weekly to determine ovarian activity from the plasma progesterone levels. A strong effect of month of parturition was found on the duration of postpartum anestrus (P < 0.0001), which was longer in females kidding in January (about 200 d) than in those kidding in May (about 100 d) or October (about 50 d). A tendency for an interaction between season of parturition and length of nursing was observed in the length of anovulation (P < 0.07): for parturition in October, anestrus was longer when kids were weaned after 90 d than after 2 or 30 d (P < 0.01). Season of parturition also affected dates of reinitiation of ovulatory and estrous activity (P < 0.001). Proportions of normal, short and long cycles and of associations between estrous and ovulations were not influenced by season of parturition or the age of weaning. These data demonstrate that in subtropical latitudes, season of parturition can dramatically influence the duration of postpartum anestrus independently of the availability of food.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

In vitro antimicrobial effect of metallic nanoparticles on phytopathogenic strains of crop plants

In this research, the in vitro antimicrobial effect of zinc oxide (ZnO), copper oxide (CuO) and iron oxide (Fe₂O₃) nanoparticles (NPs)—with average sizes of 20, 46 and 30 nm, respectively—on the root rot disease caused by the fungus Fusarium oxysporum and on blight disease caused by the fungus Alternaria solani were studied. Also, bacterial diseases caused by Clavibacter michiganensis and Pseudomonas syringae that infects a wide range of plant species were assessed. Different concentrations of NPs (0, 100, 250, 500, 700 and 1,000 mg/L) were prepared on PDA agar or King's B medium in a complete randomized design with four replicates. According to the results, ZnO NPs exhibited an outstanding inhibitory effect against fungi and bacteria strains. The above results were associated with the smaller particle size. Fungi strains showed a differential sensitivity depending on the kind of NPs used. A. solani showed the highest sensitivity to ZnO NPs at 1,000 mg/L (99%), followed by CuO NPs at the same dose (95%). Fe₂O₃ NPs at all evaluated doses had no inhibitory effects on the mycelia growth of this strain, although F. oxysporum revealed greater effectiveness of the CuO NPs (96%) compared with ZnO NPs since it only inhibited 91% of the mycelial growth. The antibacterial activity was studied through optical density. C. michiganensis was found to be more sensitive to ZnO NPs because a lesser dose (700 mg/L) was required to reduce the bacterial growth (90%); in comparison, P. syringae required a dose of 1,000 mg/L to inhibit its growth (67%). CuO NPs displayed the smallest growth inhibition against the bacteria strains analysed. The antimicrobial effect of the metallic NPs that were assayed increased with higher doses.     

Serum Lipid Levels and Dyslipidaemia Prevalence among 2–10 Year-Old Northern Mexican Children

Background and Aims

The increase in overweight and obese children may be linked to increased rates of dyslipidaemia. The aim was to assess the prevalence of dyslipidaemia and associated risk factors among the Northern Mexican child population.

Methods and Results

Four hundred and fifty-one subjects aged between 2 and 10 (47.5% girls) took part in the Nuevo León State Survey of Nutrition and Health 2011–2012. According to the 2011 Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents, serum lipid levels (mg/dL) were categorized into three subgroups (acceptable, borderline-high/low or high/low) as follows: TChol: acceptable <170, borderline-high 170–199, high ≥200; LDL-chol: acceptable <110, borderline-high 110–129, high ≥130; non-HDL-chol: acceptable <120, borderline-high 120–144, high ≥145; HDL-chol: acceptable >45, borderline-low 40–45, low <40; and TG: acceptable <75, borderline-high 75–99, high ≥100 in ≤9 year-old children, and acceptable <90, borderline-high 90–129, and high ≥130 in 10 year-old children. The overall prevalence of borderline-high + high TG, non-HDL-chol, TChol, and LDL-chol was 63.0%, 44.1%, 43.5%, and 29.9%, respectively. The overall prevalence of borderline-low + low HDL-chol was 46.3%. The overall frequency of dyslipidaemia was 54.3%. Thirteen children (2.9%) had all five symptoms of dyslipidaemia. The most common dyslipidaemia was high TG in combination (26.2%) and in isolation (10.6%).

Conclusions

Half of the children had at least one abnormal lipid concentration. A high TG level was the most frequent dyslipidaemia. Obesity was associated with the occurrence of at least one abnormal lipid level. These findings emphasize the need to pay further attention to the prevention of cardiovascular disease and obesity from an early age.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Effects of timed administration of doxycycline or methylprednisolone on post-myocardial infarction inflammation and left ventricular remodeling in the rat heart

The development of strategies to ameliorate post-myocardial infarction (MI) remodeling and improve function continues to be an area of clinical importance. Use of steroids for this purpose is controversial since the effects of timed treatment on relevant inflammatory, biochemical and structure/function endpoints are unclear. In a previous report, we demonstrated that use of doxycycline pre-treatment improves post-MI remodeling and passive left ventricular (LV) function. However, the effects of timed doxycyline post-MI treatment are unknown. To examine these issues, we performed a study using a rat MI model. Animals were administered one of the following: doxycycline (DOX), the corticosteroid methylprednisolone (MP), or aqueous vehicle. Treatment was given early, short-term (at time of MI to 24 h post-MI) or late, long term (2–7 days post-MI). Animals were sacrificed at 3, 7 or 42 days post-surgery. We assessed LV hemodynamics, pressure–volume, and pressure–scar strains, histomorphometry, inflammation via measurements of myeloperoxidase activity, and matrix metalloproteinase (MMP) activity. Late MP treatment yielded a robust right-shifted pressure–volume curve, which was accompanied by increased scar strains. Late DOX treatment yielded reduced average heart weight and size and preserved scar thickness. DOX treatment did not suppress inflammation, which contrasts with the suppressive effects of MP. Use of early or late MP yielded increased MMP activity in infarcted and non-infarcted regions. Early and late treatment with DOX yielded infarct–associated MMP activity levels comparable to those of vehicle–treated animals. In conclusion, results indicate that late use of MP yields adverse post-MI structure/function outcomes that correlate with suppression of inflammation and increased MMP activity. These observations contrast with those of DOX, in particular, late treatment where improved outcomes were observed in LV structure and were accompanied by the lack of suppression of inflammation.