The role of mitochondrial derived peptides (MDPs) in metabolism

The term "mitokines" refers to signals derived from mitochondria that have an impact on other cells or tissues (Durieux et al., 2011). Rather than being simply a set of DNA composed by 37 genes, the mitochondrial DNA (mtDNA) is quite complex and includes small RNAs (Mercer et al., 2011). Mitochondrial-derived peptides (MDPs) are encoded by functional short open reading frames (sORFs) in the mtDNA.

DNA barcoding: an effective tool to overcome morphological identification constraints in the assessment of the ecological quality

DNA barcoding has the potential to overcome taxonomic challenges in biological community assessments. However, fulfilling that potential requires successful amplification of a large and unbiased portion of the community. In this study, we attempted to identify mitochondrial gene cytochrome c oxidase I (COI) barcodes from 1024 benthic invertebrate specimens belonging to 54 taxa from low salinity environments of the Mira estuary and Torgal riverside (SW Portugal). Up to 17 primer pairs and several reaction conditions were attempted among specimens from all taxa, with amplification success defined as a single band of approximately 658 bp visualized on a pre-cast agarose gel, starting near the 5' end of the COI gene and suitable for sequencing. Amplification success was achieved for 99.6% of the 54 taxa, though no single primer was successful for more than 88.9% of the taxa. However, only 68.5% of the specimens within these taxa successfully amplified. Inhibition factors resulting from a non-purified DNA extracted and inexistence of species-specific primers for COI were pointed as the main reasons for an unsuccessful amplification. These results suggest that DNA barcoding can be an effective tool for application in low salinity environments where taxa such as chironomids and oligochaetes are challenging for morphological identification. Nevertheless, its implementation is not simple, as methods are still being standardized and multiple species

Mitochondrial demographic history of the Egyptian mongoose (Herpestes ichneumon), an expanding carnivore in the Iberian Peninsula

Describing the genetic patterns and the demographic history of expanding species is essential for providing insights into the processes linked with range dynamics. We analysed the mitochondrial diversity of the Egyptian mongoose (Herpestes ichneumon) across the Iberian Peninsula, where the species is currently expanding northwest. A total of 242 individuals were analysed, together with nine representatives from the North African dispersal source. Haplotype segregation and strong differentiation between Iberian and North African populations confirmed the longterm presence of the species in the Iberian Peninsula. The distribution of mitochondrial diversity fitted the pattern of a historically diversified population in southern Iberia, from which the recent dispersals into northern areas may have occurred. Higher levels of haplotype and nucleotide diversities in the northern areas, together with the heterogeneous distribution of pairwise population differentiations and the weak signal for isolationbydistance suggest the existence of longdispersal migrants across the Iberian Peninsula. Sudden and spatial expansion scenarios of H. ichneumon in the Iberian Peninsula were supported by mismatch analysis and marginally supported by neutrality tests. However, the precise time of occurrence of the detected expansion remains unclear. Future studies should incorporate additional markers in order to further clarify the population dynamics of the Egyptian mongoose in its Iberian range.

R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features.

Inhibition and characterization of two-metal ion enzymes to treat cancer: dna polymerase Ƞ and topoisomerase II α

Chemotherapeutic drugs can in many ways disrupt the replication machinery triggering apoptosis in cancer cells: some act directly on DNA and others block the enzymes involved in preparing DNA for replication. Cisplatin-based drugs are common as first-line cancer chemotherapics. Another example is etoposide, a molecule that blocks topoisomerase II α leading to the inhibition of dsDNA replication. Despite their efficacy, cancer cells can respond to these treatments over time by overtaking their effects, leading to drug resistance. Chemoresistance events can be triggered by the action of enzymes like DNA polymerase ƞ (Pol η). This polymerase helps also to bypass drug-induced damage in cancer cells, allowing DNA replication and cancer cells proliferation even when cisplatin-based chemotherapeutic drugs are in use. Pol ƞ is a promising drug discovery target, whose inhibition would help in overcoming of drug resistance. This study aims to identify a potent and selective Pol ƞ inhibitor able to improve the efficacy of platinum-based chemotherapeutic drugs. We report the discovery of compound 64 (ARN24964), after an extensive SAR reporting 35 analogs. We evaluated compound 64 on four different cell lines. Interestingly, the molecule is a Pol η inhibitor able to act synergistically with cisplatin. Moreover, we also synthesized a prodrug form that allowed us to improve its stability and the bioavailability. This compound represents an advanced scaffold featuring good potency and DMPK properties. In addition to this central theme, this thesis also describes our efforts in developing and characterize a novel hybrid inhibitor/poison for the human topoisomerase II α enzyme. In particular, we performed specific assays to study the inhibiton of Topoisomesare II α and we evaluated compounds effect on three cancer cell lines. These studies allowed us to identify a compound that is able to inhibit the enzyme with a good pK and a good potency.

Interplay Between Cell Growth And Cell Cycle In Plants.

The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Biological activity of metal-edds (ethylenediaminedisuccinate) complexes in K562 and PBMC cells

The effect of S,S-ethylenediaminedisuccinic acid (edds) on the quenching of metal-catalyzed (metal = Mn, Fe, Co, Ni, Cu, Zn) oxidation of ascorbic acid was tested in vitro via oxidation of the fluorescent probe 1,2,3-dihydrorhodamine dihydrochloride. The pro-oxidant activity of iron was not fully suppressed, even at a four-fold molar excess of the ligand. The effect of serum on the toxicity to peripheral blood mononuclear cells (PBMC) and K562 cells was investigated. The cytotoxic effect of Fe-edds was abrogated in the presence of Trolox or serum proteins. The probable pathways of cell toxicity were investigated through blocking of the monocarboxylate transporters (MCT) in association with cell cycle studies by flow cytometry. Cells treated with metal complexes and alpha-cyano-4-hydroxycinnamic acid, a known MCT inhibitor, showed recovery of viability, suggesting that MCT proteins may be involved in the internalization of metal-edds complexes. The free acid induced cell cycle arrest in G0/G1 (PBMC) and S (K562) phases, suggesting direct DNA damage or interference in DNA replication.

Morphometric and genetic changes in a population of Apis mellifera after 34 years of Africanization

Though the replacement of European bees by Africanized honey bees in tropical America has attracted considerable attention, little is known about the temporal changes in morphological and genetic characteristics in these bee populations. We examined the changes in the morphometric and genetic profiles of an Africanized honey bee population collected near where the original African swarms escaped, after 34 years of Africanization. Workers from colonies sampled in 1968 and in 2002 were morphometrically analyzed using relative warps analysis and an Automatic Bee Identification System (ABIS). All the colonies had their mitochondrial DNA identified. The subspecies that mixed to form the Africanized honey bees were used as a comparison for the morphometric analysis. The two morphometric approaches showed great similarity of Africanized bees with the African subspecies, Apis mellifera scutellata, corroborating with other markers. We also found the population of 1968 to have the pattern of wing venation to be more similar to A. m. scutellata than the current population. The mitochondrial DNA of European origin, which was very common in the 1968 population, was not found in the current population, indicating selective pressure replacing the European with the African genome in this tropical region. Both morphometric methodologies were very effective in discriminating the A. mellifera groups; the non-linear analysis of ABIS was the most successful in identifying the bees, with more than 94% correct classifications.

Genetic variability of the Amazon River prawn Macrobrachium amazonicum (Decapoda, Caridea, Palaemonidae)

The freshwater prawn Macrobrachium amazonicum is widely distributed in South America, and occupies habitats with a wide range of salinities. Several investigations have revealed the existence of wide intraspecific variability among different populations, although the understanding of this variability is still fragmentary and incomplete. We compared and characterized inland and coastal populations of M. amazonicum from Brazil, using molecular data (16S and COI mtDNA) to describe the degree of variability, structure, and relationships among them. Genetic divergence rates among populations showed variability at the intraspecific level. All the analyses evidenced significant genetic divergence among populations, structuring them in three groups: I-inland waters of the Amazonian Hydrographic Region (HR); II-Parana/Paraguay HR; and III-coastal systems of northern and northeastern Brazil. Phylogenetic reconstructions revealed that the populations form a single monophyletic clade, which supports their characterization as a single species. Clade I was a sister clade of that formed by clades II and III, which were themselves sister clades. Populations from Sertaozinho/Miguelopolis and Avare, introduced into the state of Sao Paulo, may have originated from natural populations in the states of Mato Grosso do Sul and Para, respectively. Geographical isolation probably contributed to the observed variation, and if this isolation continues. M. amazonicum may undergo speciation within its broad geographical distribution. The sequences obtained here can be used as name-tags for population identification, and the DNA barcodes are useful to identify the origin of specimens used in different freshwater-prawn cultures or introduced populations of unknown origin.

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil

Background: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The ""Folmer region"" detects a single taxon using a 3% divergence threshold. Methods: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2). Results: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (< 100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (similar to 798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapa state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage. Conclusions: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Inferences of evolutionary and ecological events that influenced the population structure of Plebeia remota, a stingless bee from Brazil

The present study characterised the population genetic structure of Plebeia remota through mitochondrial DNA (mtDNA) analysis and evaluated evolutionary and ecological processes that may have contributed to the species current genetic scenario. Seventy feral nests were sampled representing four geographic regions (Cunha, Curitiba, Prudentopolis, and Blumenau). Fifteen composite mtDNA haplotypes were determined and a high genetic structure was detected among all populations. The current population structure may be a result of queen philopatry and vegetation shifts caused by palaeoclimatic changes and uplift of Brazilian coastal ranges. Finally, this study strongly suggests a revision of the taxonomic status of P. remota from the Prudentopolis region.

Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra-and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

Industrial apiculture in the Jordan valley during Biblical times with Anatolian honeybees

Although texts and wall paintings suggest that bees were kept in the Ancient Near East for the production of precious wax and honey, archaeological evidence for beekeeping has never been found. The Biblical term ""honey"" commonly was interpreted as the sweet product of fruits, such as dates and figs. The recent discovery of unfired clay cylinders similar to traditional hives still used in the Near East at the site of Tel Rehov in the Jordan valley in northern Israel suggests that a large-scale apiary was located inside the town, dating to the 10th-early 9th centuries B.C.E. This paper reports the discovery of remains of honeybee workers, drones, pupae, and larvae inside these hives. The exceptional preservation of these remains provides unequivocal identification of the clay cylinders as the most ancient beehives yet found. Morphometric analyses indicate that these bees differ from the local subspecies Apis mellifera syriaca and from all subspecies other than A. m. anatoliaca, which presently resides in parts of Turkey. This finding suggests either that the Western honeybee subspecies distribution has undergone rapid change during the last 3,000 years or that the ancient inhabitants of Tel Rehov imported bees superior to the local bees in terms of their milder temper and improved honey yield.