Developmental staging table of the green iguana

Embryonic staging tables provide information to standardize embryological investigations and to subsidize discussions about evolution. We have established a developmental staging table for Iguana iguana iguana. The sample was composed of 142 embryos, incubated at a constant temperature and collected at regular intervals. Morphological features as pharyngeal arches, craniofacial structures, eyes, limbs, claws, pigmentation, scales and egg tooth were evaluated to determine development stages. The normal staging table includes 17 stages from oviposition to hatching, based on chronology and morphological external features. Stages from 1 to 27 occur before oviposition. Stage 28 was the first described, because all embryos presented limb bud anlage, key feature of the previous stage. We used pharyngeal arches and limb buds to describe the first stages; claws, genital papilla and scales to describe the middle stages; and pigmentation, size and egg tooth to describe the last stages. Incubation lasted approximately 2 months in a controlled environment. The results were similar to the data from other lizards, confirming the embryonic conservative pattern of the group.     

Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases

Oxidative damage represents a major threat to genomic stability, as the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. In order to prevent these mutagenic events, organisms have evolved GO-DNA glycosylases that remove this oxidized base from DNA. We were interested to find out how GO is processed in the hyperthermophilic archaeon Pyrobaculum aerophilum, which lives at temperatures around 100°C. To this end, we searched its genome for open reading frames (ORFs) bearing the principal hallmark of GO-DNA glycosylases: a helix–hairpin–helix motif and a glycine/proline-rich sequence followed by an absolutely conserved aspartate (HhH-GPD motif). Interestingly, although the P.aerophilum genome encodes three such ORFs, none of these encodes the potent GO-processing activity detected in P.aerophilum extracts. Fractionation of the extracts, followed by analysis of the active fractions by denaturing polyacrylamide gel electrophoresis, showed that the GO-processing enzyme has a molecular size of ~30 kDa. Mass spectrometric analysis of proteins in this size range identified several peptides originating from P.aerophilum ORF PAE2237. We now show that PAE2237 encodes AGOG (Archaeal GO-Glycosylase), the founding member of a new family of DNA glycosylases, which can remove GO from single- and double-stranded substrates with great efficiency.     

Antimicrobial activity of fractions and compounds from Calophyllumbrasiliense (Clusiaceae/Guttiferae)

Calophyllum brasiliense (Clusiaceae/Guttiferae) is a native Brazilian medicinal plant traditionally used against several diseases, including infectious pathologies. Crude methanolic extracts (CME) and two fractions, denoted non-polar (soluble in chloroform) and polar (nonsoluble in chloroform), were prepared from different parts of the plant (roots, stems, leaves, flowers and fruits) and studied. The following compounds were isolated and tested against pathogenic bacteria and yeasts by determination of the minimal inhibitory concentration (MIC): brasiliensic acid (1), gallic acid (2), epicatechin (3), protocatechuic acid (4), friedelin (5) and 1,5-dihydroxyxanthone (6). The results indicated that all the parts of the plant exhibited antimicrobial activity against Gram-positive bacteria, which are selectively inhibited by components of C. brasiliense. No activity was observed against Gram-negative bacteria and yeasts tested. Regarding the isolated compounds, substance 4 showed antimicrobial activity against all the tested microorganisms, whereas compound 6 exhibited antimicrobial activity only against Gram-positive bacteria. The results from the current study confirm and justify the popular use of this plant to treat infectious processes.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate

It has been shown that neutral-sphingomyelinase and sphingomyelin-synthase activities are present in chromatin and they modify the sphingomyelin (SM) content. The activity of the first enzyme is stimulated and the second inhibited, when the hepatocytes enter into the S-phase after partial hepatectomy, thus suggesting that ceramide may have a pivotal role in cell proliferation. An opposite function was attributed to ceramide in hepatocytes which undergo apoptosis after lobular ligature. In order to clarify this point, a model was developed in which the same liver cells undergo proliferation followed by induced apoptosis. To this purpose, the rats were treated for 7 days with ciprofibrate and then left without treatment for 4 days. During the treatment, the peroxisome enzyme markers increase their activity and the number of proliferating cells increases, reaching a maximum after 3 days of treatment, as shown by the number of cells positive for the proliferating cell nuclear antigen. At the same time, the chromatin sphingomyelinase activity reaches the maximum, while a similar increase is not found in the cytoplasm or in the isolated nuclei. On the contrary, SM-synthase activity is depressed in chromatin, but not in the nuclei in which a peak is shown after 3 days of ciprofibrate treatment. After drug withdrawal, the hepatocytes undergo apoptosis as confirmed by the increase of Bax and tissue transglutaminase (tTGase) expression; the chromatin SM increases as a consequence of an increase of SM-synthase activity. It can be hypothesised that chromatin SM may have a role in cell duplication by influencing the chromatin structure stability.     

Acidophilic character of yeast PID261/BUD32, a putative ancestor of eukaryotic protein kinases

Yeast piD261/Bud32 and its homologues are present in eukaryotes and in archaea but not in bacteria and are believed to make up a primordial branch of the eukaryotic protein kinase superfamily. Here, we show that, at variance with the majority of Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic and/or proline residues, piD261 phosphorylates in vitro a number of acidic proteins and peptides, and it recognizes seryl residues specified by carboxylic side chains. These data suggest that recognition of acidic sites might have been a primordial trait of protein kinases, which was modified during evolution to cope with the increasing complexity of protein phosphorylation in eukaryotes.     

Echocardiographic and invasive measurements of pulmonary artery pressure correlate closely at high altitude

Exaggerated hypoxia-induced pulmonary hypertension is a hallmark of high-altitude pulmonary edema (HAPE) and plays a major role in its pathogenesis. Many studies of HAPE have estimated systolic pulmonary arterial pressure (SPAP) with Doppler echocardiography. Whereas at low altitude, Doppler echocardiographic estimation of SPAP correlates closely with its invasive measurement, no such evidence exists for estimations obtained at high altitude, where alterations of blood viscosity may invalidate the simplified Bernoulli equation. We measured SPAP by Doppler echocardiography and invasively in 14 mountaineers prone to HAPE and in 14 mountaineers resistant to this condition at 4,559 m. Mountaineers prone to HAPE had more pronounced pulmonary hypertension (57 +/- 12 and 58 +/- 10 mmHg for noninvasive and invasive determination, respectively; means +/- SD) than subjects resistant to HAPE (37 +/- 8 and 37 +/- 6 mmHg, respectively), and the values measured in the two groups as a whole covered a wide range of pulmonary arterial pressures (30-83 mmHg). Spearman test showed a highly significant correlation (r = 0.89, P < 0.0001) between estimated and invasively measured SPAP values. The mean difference between invasively measured and Doppler-estimated SPAP was 0.5 +/- 8 mmHg. At high altitude, estimation of SPAP by Doppler echocardiography is an accurate and reproducible method that correlates closely with its invasive measurement.     

Cost-Effectiveness Analysis of Universal Vaccination of Adults Aged 60 Years with 23-Valent Pneumococcal Polysaccharide Vaccine versus Current Practice in Brazil

Objective

To evaluate the cost-effectiveness of introducing universal vaccination of adults aged 60 years with the 23-valent pneumococcal polysaccharide vaccine (PPV23) into the National Immunization Program (NIP) in Brazil.

Methods

Economic evaluation using a Markov model to compare two strategies: (1) universal vaccination of adults aged 60 years with one dose of PPV23 and 2) current practice (vaccination of institutionalized elderly and elderly with underlying diseases). The perspective was from the health system and society. Temporal horizon was 10 years. Discount rate of 5% was applied to costs and benefits. Clinical syndromes of interest were invasive pneumococcal disease (IPD) including meningitis, sepsis and others and pneumonia. Vaccine efficacy against IPD was obtained from a meta-analysis of randomized control trials and randomized studies, whereas vaccine effectiveness against pneumonia was obtained from cohort studies. Resource utilization and costs were obtained from the Brazilian Health Information Systems. The primary outcome was cost per life year saved (LYS). Univariate and multivariate sensitivity analysis were performed.

Results

The universal vaccination strategy avoided 7,810 hospitalizations and 514 deaths, saving 3,787 years of life and costing a total of USD$31,507,012 and USD$44,548,180, respectively, from the health system and societal perspective. The universal immunization would result in ICERs of USD$1,297 per LYS, from the perspective of the health system, and USD$904 per LYS, from the societal perspective.

Conclusion

The results suggest that universal vaccination of adults aged 60 years with the 23-valent pneumococcal polysaccharide vaccine (PPV23) is a very cost-effective intervention for preventing hospitalization and deaths for IPD and pneumonia is this age group in Brazil.     

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.     

Neuroendocrine immunity in patients with Alzheimer's disease: toward translational epigenetics

The emerging domain of epigenetics in molecular medicine finds application for a variety of patient populations. Here, we present fundamental neuroendocrine immune evidence obtained in patients with senile dementia of the Alzheimer's type (sDAT), and discuss the implications of these data from the viewpoint of translational epigenetics of Alzheimer's disease. We followed 18 subjects with mild sDAT treated with acetylcholinesterase inhibitors, and 10 control subjects matched for age in a repeated measure design every six months for 18 months. We monitored psychosocial profile (Mini-Mental State Examination, Functional Assessment Staging, Independence in Activities of Daily Living, Depression, Profile of Moods States) in parallel to immunophenotypic parameters of T cell subpopulations by flow cytometry. Based on change in the mini-mental state score at entry and at 18 months, patients with sDAT were assigned to a "fast progression" (delta greater than 2 points) or to a "slow progression" group (delta less than or equal to 2 points). The change in circulating activated T cells (CD3+Dr+) with time in patients with sDAT was significantly inversely correlated with the change in time in natural killer (NK) cytotoxic activity to cortisol modulation in these patients, which was greater in patients with fast progression, compared to slow progression sDAT. These data indicate underlying neuroendocrine immune processes during progression of sDAT. Our observations suggest that psychoimmune measures such as those we have monitored in this study provide relevant information about the evolving physiological modulation in patients with sDAT during progression of Alzheimer's disease, and point to new or improved translational epigenetic treatment interventions.