Genetic Variation among Major Human Geographic Groups Supports a Peculiar Evolutionary Trend in PAX9

A total of 172 persons from nine South Amerindian, three African and one Eskimo populations were studied in relation to the Paired box gene 9 (PAX9) exon 3 (138 base pairs) as well as its 5' and 3' flanking intronic segments (232 bp and 220 bp, respectively) and integrated with the information available for the same genetic region from individuals of different geographical origins. Nine mutations were scored in exon 3 and six in its flanking regions; four of them are new South American tribe-specific singletons. Exon3 nucleotide diversity is several orders of magnitude higher than its intronic regions. Additionally, a set of variants in the PAX9 and 101 other genes related with dentition can define at least some dental morphological differences between Sub-Saharan Africans and non-Africans, probably associated with adaptations after the modern human exodus from Africa. Exon 3 of PAX9 could be a good molecular example of how evolvability works.

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

Low urinary 6-sulphatoxymelatonin concentrations in acute migraine

Substantial evidence points to melatonin as playing a role in the regulation of circadian rhythms, sleep, and headache disorders. The objective of the study was to assess 6-sulphatoxymelatonin (aMT6s) levels in a large consecutive series of patients with migraine, comparing with controls. A total of 220 subjects were evaluated-146 had migraine and 74 were control subjects. Urinary samples were collected into the same plastic container since 8:00 p.m. to 8:00 a.m. of the next day (12-h period) and aMT6s was measured with quantitative ELISA technique. Among patients with migraine, 53% presented pain on the day of the urine samples collection. Their urinary aMT6s concentration was significantly lower than in the urine of patients without pain [14.0 +/- 7.3 vs. 49.4 +/- 19.0; t(143) = -15.1; 95% CI = -40.0 to -30.8; P<0.001]. There was no significant difference in the aMT6s concentration of patients with migraine without pain on the day of their urine samples collection and controls [49.4 +/- 19.0 vs. 42.5 +/- 27.9; t(140) = 1.7; 95% CI = -1.2 to 14.8; P = 0.094]. To our knowledge, this is the first study to demonstrate reduction in melatonin levels during attacks in episodic and chronic migraine.

Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged alpha-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Genome Size, Karyotype Polymorphism and Chromosomal Evolution in Trypanosoma cruzi

Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites. Methodology/Principal Findings: The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (Jose-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively. Conclusions: Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

Supramolecular polymorphism of DNA in non-cationic L(alpha) lipid phases

The structure of a complex between hydrated DNA and a non-cationic lipid is studied, including its phase diagram. The complex is spontaneously formed by adding DNA fragments (ca. 150 base pairs in length) to non-cationic lipids and water. The self-assembly process often leads to highly ordered structures. The structures were studied by combining X-ray scattering, fluorescence and polarized microscopy, as well as freeze-fracture experiments with transmission electron microscopy. We observe a significant increase of the smectic order as DNA is incorporated into the water layers of the lamellar host phase, and stabilization of single phase domains for large amounts of DNA. The effect of confinement on DNA ordering is investigated by varying the water content, following three dilution lines. A rich polymorphism is found, ranging from weakly correlated DNA-DNA in-plane organizations to highly ordered structures, where transmembrane correlations lead to the formation of columnar rectangular and columnar hexagonal superlattices of nucleotides embedded between lipid lamellae. From these observations, we suggest that addition of DNA to the lamellar phase significantly restricts membrane fluctuations above a certain concentration and helps the formation of the lipoplex. The alteration of membrane steric interactions, together with the appearance of interfacial interactions between membranes and DNA molecules may be a relevant mechanism for the emergence of highly ordered structures in the concentrated regime.

Electron collisions with the CH(2)O-H(2)O complex

We report cross sections for elastic collisions of low-energy electrons with the CH(2)O-H(2)O complex. We employed the Schwinger multichannel method with pseudopotentials in the static-exchange and in the static-exchange-polarization approximations for energies from 0.1 to 20 eV. We considered four different hydrogen-bonded structures for the complex that were generated by classical Monte Carlo simulations. Our aim is to investigate the effect of the water molecule on the pi* shape resonance of formaldehyde. Previous studies reported a pi* shape resonance for CH(2)O at around 1 eV. The resonance positions of the complexes appear at lower energies in all cases due to the mutual polarization between the two molecules. This indicates that the presence of water may favor dissociation by electron impact and may lead to an important effect on strand breaking in wet DNA by electron impact.

Comparison of (120)Sn((6)He, (6)He)(120)Sn and (120)Sn(alpha,alpha)(120)Sn elastic scattering and signatures of the (6)He neutron halo in the optical potential

Cross sections of (120)Sn(alpha,alpha)(120)Sn elastic scattering have been extracted from the alpha-particle-beam contamination of a recent (120)Sn((6)He,(6)He)(120)Sn experiment. Both reactions are analyzed using systematic double-folding potentials in the real part and smoothly varying Woods-Saxon potentials in the imaginary part. The potential extracted from the (120)Sn((6)He,(6)He)(120)Sn data may be used as the basis for the construction of a simple global (6)He optical potential. The comparison of the (6)He and alpha data shows that the halo nature of the (6)He nucleus leads to a clear signature in the reflexion coefficients eta(L) : The relevant angular momenta L with eta(L) >> 0 and eta(L) << 1 are shifted to larger L with a broader distribution. This signature is not present in the alpha-scattering data and can thus be used as a new criterion for the definition of a halo nucleus.

alpha-particle production in (6)He+(120)Sn collisions

The collision (6)He+ (120)Sn has been investigated at four energies near the Coulomb barrier. A large yield of a particles has been detected, with energies around the energy of the scattered (6)He beam. The energy and angular distributions of the a particles have been analyzed and compared with breakup and neutron transfer calculations.

Elastic scattering and total reaction cross section of (6)He+(120)Sn

The elastic scattering of (6)He on (120)Sn has been measured at four energies above the Coulomb barrier using the (6)He beam produced at the RIBRAS (Radioactive Ion Beams in Brasil) facility. The elastic angular distributions have been analyzed with the optical model and three- and four-body continuum-discretized coupled-channels calculations. The total reaction cross sections have been derived and compared with other systems of similar masses.

Breakup coupling effects on near-barrier quasi-elastic scattering of (6,7)Li on (144)Sm

Excitation functions of quasi-elastic scattering at backward angles have been measured for the (6,7)Li + (144)Sm systems at near-barrier energies, and fusion barrier distributions have been extracted from the first derivatives of the experimental cross sections with respect to the bombarding energies. The data have been analyzed in the framework of continuum discretized coupled-channel calculations, and the results have been obtained in terms of the influence exerted by the inclusion of different reaction channels, with emphasis on the role played by the projectile breakup.