Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors

Copyright © 2020 American Chemical Society. The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding

A novel Modulator of Ring Stage Translation (MRST) gene alters artemisinin sensitivity in Plasmodium falciparum

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum. In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7_1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite’s ART drug response

Chemogenomic profiling of a Plasmodium falciparum transposon mutant library reveals shared effects of dihydroartemisinin and bortezomib on lipid metabolism and exported proteins

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance

Phenotypic screens identify genetic factors associated with gametocyte development in the human malaria parasite Plasmodium falciparum

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen

Mitochondrial DNA sequence-based phylogeny of Melipona honeybee Rufiventris complex

O complexo de abelhas rufiventris, da tribo Meliponini, apresenta ampla distribuição no território nacional. Com uma extensa distribuição geográfica, é possível que este grupo de abelhas apresente ecótipos diferentes, adaptados a diferentes locais. A identificação de espécies do gênero Melipona por caracteres morfológicos não é fácil. Embora M. mondury e M. rufiventris tenha sido diferenciada morfologicamente por, o mesmo autor relata a observação de características de ambas as espécies em indivíduos de algumas localidades dos estados de Goiás, Maranhão, Ceará e Rio Grande do Norte. Trabalhos com diferentes técnicas moleculares (PCR-RFLP, PCR-RAPD, Microssatelite, PCR-ISSR, Isoenzimas e sequenciamento de DNAmt) mostraram que a maioria dos indivíduos do complexo rufiventris analisados (do noroeste de Minas ao Maranhão) não se inclui em nenhum dos padrões moleculares descritos para M. rufiventris e M. mondury. Estes indivíduos pertenceriam a uma nova espécie? Considerando a existência de questões ainda não resolvidas dentro do complexo rufiventris, propõe-se, no presente estudo, a utilização de ferramentas adicionais, como análises filogenéticas, que forneçam dados para uma discussão mais ampla a respeito da distribuição geográfica no território brasileiro, da diversidade genética e taxonomia de espécies deste complexo. Operárias de abelhas do complexo rufiventris foram amostradas em diferentes localidades dos estados de Minas Gerais, Goiás, Maranhão, Ceará, Piauí e Tocantins. O DNA total foi extraído, a região COICOII do DNAmt dessas abelhas foram amplificadas, as sequências de DNA obtidas foram purificadas, clonadas, os plamídeos foram extraídos e os respectivos insertos foram sequenciados. O gene cytb também do DNAmt, foi amplificado, os fragmentos de DNA foram purificados e diretamente sequenciados. As sequências foram editadas e alinhadas utilizando softwares apropriados. Em seguida as sequências obtidas foram concatenadas, haplótipos foram identificados e analisados filogeneticamente. Árvores filogenéticas foram construídas por máxima verossimilhança e inferência bayesiana. Para melhor detalhamento dos resultados, redes de haplótipos foram construídas com base nas sequências de DNA obtidas. As relações filogenéticas inferidas resultaram em uma árvore com quatro clados bem definidos: M. mondury, M. rufiventris, M. flavolineata e Melipona sp., formando dois grupos de espécies irmãs: M. mondury e M. rufiventris, e M. flavolineata e Melipona sp.. As redes de haplótipos obtidas sugerem que o ancestral do grupo formado por M. flavolineata e Melipona sp. viveu em uma região entre os estados do Maranhão, Piauí e Ceará e que parece existir algum evento influenciando a separação dos haplótipos identificados no clado M. rufiventris dentro da área de amostragem. Os resultados obtidos neste estudo sugerem, também, a existência de uma espécie pertencente ao complexo rufiventris ainda não descrita (Melipona sp.) e com ampla distribuição geográfica. Estudos mais detalhados são necessários visando elucidar questões não respondidas no presente estudo, dentre outras.The rufiventris bee complex, tribe Meliponini, is widely distributed within the Brazil. With an extensive geographic distribution, this group may present different ecotypes adapted to different locations. The identification of species of the genus Melipona by morphological characters is not easy. Reported some morphological differences between M. mondury and M. rufiventris, however in individuals of some locations (Goiás, Maranhão, Ceará and Rio Grande do Norte), were found characters of both species. Studies with different molecular techniques (PCR-RFLP, PCR-RAPD, microsatellites, ISSR-PCR, Isoenzyme and mtDNA sequencing) showed that most individuals of rufiventris complex examined (north-west of Minas to Maranhão) is not included in any of the patterns described for M. rufiventris and M. mondury. Do these individuals belong to a new specie? Due to the problems related with this genus, it was proposed the use of new tools that provide useful data for a wider discussion about the geographical distribution in the brazilian territory, genetic diversity and taxonomy of the rufiventris. Thus, evaluating rufiventris complex species based on phylogenetic analysis can help to better understand taxonomic issues that still remain unsolved. Worker honeybees of the rufiventris complex were sampled in different locations of the states of Minas Gerais, Goiás, Maranhão, Ceará, Piauí and Tocantins. Total DNA was extracted, the COICOII region of mtDNA of these bees were amplified, purified, and the cloned vectors were extracted and sequenced. The cytb gene of mtDNA was also amplified, purified and sequenced. The sequences were edited and aligned using appropriate softwares. After, the sequences of both genes were concatenated generating haplotypes and these were phylogenetically analyzed, generating trees. These trees were constructed by maximum likelihood and Bayesian inference. In addition, a network of haplotypes was obtained for more detail of the results. The inferred phylogenetic relationships resulted in a tree with four well-defined clades: M. mondury, M. rufiventris, M. flavolineata and Melipona sp., forming two groups of sister species: M. mondury and M. rufiventris, M. flavolineata and Melipona sp.. The haplotypes networks obtained suggest that the ancestor of the group formed by M. flavolineata and Melipona sp. lived in the region between the states of Maranhão, Piauí and Ceará and there is some event influencing the separation of the haplotypes identified in the M. rufiventris clade within the sampling area. The results of this present study also suggest the existence of a specie, still undescribed (Melipona sp.), belonging to the rufiventris complex which presents a wide geographical distribution. More detailed studies are needed to elucidate unsolved questions in the present study, among others.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Gene regulation of early developmental processes of haploid and diploid embryos of Apis mellifera

O desenvolvimento embrionário é o resultado de uma sequência controlada de eventos modulados por sinais ambientais e mecanismos intracelulares. Em Hymenoptera, esse processo tem um caráter especial devido ao sistema de determinação do sexo (Haplodiploide). Neste sistema, os ovos fecundados se desenvolvem em fêmeas (diploides) e os ovos não fecundados em machos (haploides). Assim, eventos importantes, como a ativação do ovo e transição materno-zigótica, eventos iniciais da embriogênese, são elementos-chave para compreender o desenvolvimento de ambos os tipos de embriões. Ativação do ovo é um evento complexo acionado em resposta a estímulos externos, necessários para o início da embriogênese. Em abelhas a ativação ovo ocorre independentemente da fecundação e parece ser desencadeado durante a passagem pelo trato reprodutivo da mãe. Além disso, se o ovócito não for fecundado ele irá se desenvolver em um organismo haploide. No entanto, se o ovo recebe o espermatozóide até 30 minutos depois da ativação, o ovo se desenvolve em um organismo diploide. Em Drosophila, a ativação do ovo é também idependente da fecundação. O estímulo inicial que desencadeia o desenvolvimento é devido tensões mecânicas sofridas pelo ovócito durante a ovulação pela passagem através do trato reprodutivo. Neste modelo, o primeiro sinal de ativação inclui a ativação da via dependente de cálcio. Moléculas maternas que são incorporados no ovócito durante ovogênese, atuam durante a ativação do ovo, bem como no início da embriogênese. Os eventos iniciais da embriogênese também são caracterizados pela ausência de altos níveis de transcrição zigótica. As moléculas depositadas atuam na ativação do ovo, quebrando a dormência da divisão celular permitindo a ocorrência do início do desenvolvimento embrionário. Mas, o embrião em desenvolvimento gradualmente degrada e substitui essas moléculas herdadas da mãe, em um processo conhecido como transição materno-zigótica. Nosso principal objetivo foi o entendimento da comunicação entre as moléculas herdadas e as recém produzidas durante os primeiros passos do desenvolvimento de Apis mellifera. Para alcançar nosso objetivo, 16 bibliotecas de RNAseq (mRNA e miRNA) foram construídas utilizando amostras de RNA total de embriões diploides e haploides de diferentes idades e ovócitos maduros. A análise do transcriptoma mostrou que existem genes diferencialmente expressos entre os dois tipos de embriões já em 1 h de desenvolvimento. Além disso, nossa análise permitiu a identificação de mRNAs e miRNAs maternos e zigóticos, além de processos com que estas moléculas se relacionam. As análises mostraram também que um mesmo miRNA pode atingir diferentes mRNAs em cada tipo de embrião, na mesma fase de desenvolvimento. Além disso, um mesmo gene pode ser diferentemente regulado nos dois tipos de embriões. Por exemplo, broad/GB48272, que é classificado como materno em embriões dipoides é regulado por quatro miRNAs diferentes e em embriões haploides é classificado como zigótico, regulado por apenas um miRNA. Análise das bibliotecas de RNAseq e hibridação in situ mostrou o padrão de expressão de zelda em embriões jovens de abelhas. Zelda é um ativador chave do genoma zigótico em Drosophila e regula eventos importantes na embriogênese se ligando a um motivo conservado, TAGteam. Em A. mellifera, encontramos um motivo TAGteam putativo que tem sido relacionado à transcrição zigótica precoce. Além disso, a hibridização in situ e PCR mostraram três primiRNAs (ame-mir-375-3p, ame-mir-34-5p e ame-mir-263b-5p) que se expressam durante a clivagem. A presença de pri-miRNAs evidenciou a início da transcrição zigótica durante a clivagem. Em suma, podemos dizer que este é o primeiro trabalho em Apis mellifera a descrever os eventos de iniciais do desenvolvimento embrionário comparando embriões haploides e diploides usando os recentes protocolos de bioinformática e os avanços da biologia molecular.Embryonic development is the result of a precisely controlled sequence of events modulated by environmental signals and intracellular mechanisms. In Hymenoptera, this process takes a special character due the sex-determination system (haplodiploidy). In this system, fertilized eggs develop in females (diploid) and unfertilized eggs in males (haploid). Thus, important events such as egg activation and maternal-zygotic transition, events of the early embryogenesis are key elements to understand the development of both types of embryos. Egg activation is a complex event triggered in response to external stimuli and necessary for the onset of embryogenesis. In honeybees egg activation occurs independently of fertilization and seems to be triggered during the passage through mother\'s reproductive tract. Furthermore, if the egg is not fertilized it will develop into haploid organism. However, if the egg receives the sperm up to 30min after activation, this egg develops into a diploid organism. In Drosophila, the egg activation is also fertilization independent. Initial stimulus that triggers the development is due mechanical stresses suffered by the egg during ovulation and passage through the reproductive tract. In this model, the first activation signal includes activation of calciumdependent pathway. Maternal molecules that are incorporated into the oocyte during ovogenesis, act during egg activation, as well as in early embryogenesis. Early embryogenesis events are also characterized by absence of high levels of zygotic transcription. The deposited molecules drive egg activation, breaking cell division dormancy permitting the beginning of embryonic development. But, the developing embryo gradually degrades and substitutes these mother-inherited molecules, in a process known as mother-to-zygote transition. Our main objective was the understanding of the deep crosstalk among the inherited molecules and the newly ones produced during the first steps of Apis mellifera embryogenesis. To achieve our objective 16 deep sequenced RNA (mRNA, miRNA) libraries were constructed using different age diploid and haploid embryos, and mature oocytes. Genome-wide transcriptome analysis was performed and interactive regulatory networks were constructed. Our analysis permitted the identification of maternal and zygotic mRNAs and miRNAs and related processes. Based on expression profiles of mRNAs and miRNAs in mature oocytes and haploid and diploid embryos of 2, 6 and 18-24 h of development, we constructed integrative regulatory networks (miRNA:mRNA) showing that the same miRNA could target different mRNAs in each type of embryo, in the same phase of development. As example we cite broad/GB48272, which is classified as maternal in diploid embryos and regulated by four different miRNAs. However, in haploid embryos it is zygotic and regulated by only one miRNA. Analysis of RNAseq and in situ hybridization showed the expression pattern of zelda in early honeybee embryos. Zelda is a key activator of Drosophila early zygotic genome and regulates important events in early embryogenesis binding to TAGteam motif. In A. mellifera, we found a putative TAGteam motif that has been implicated in early zygotic transcription. Moreover, in situ hybridization and PCR assay showed three pri-miRNAs (ame-mir-375-3p, ame-mir-34-5p and ame-mir-263b-5p) expressed during cleavage. The presence of pri-miRNAs is the first evidence of early zygotic transcription during cleavage. In short, we could say that this is the first work on Apis mellifera describing the early embryonic developmental events comparing haploid and diploid embryos using modern bioinformatics tools and advanced molecular analysis

Oxidative stress changes the effectiveness of artemisinin in Plasmodium falciparum

ABSTRACTMalaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite’s tolerance of the activated drug’s oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite’s adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite’s survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite’s response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin.IMPORTANCEEmerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite’s responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients

Transcriptome analysis of honeybee (Apis Mellifera) haploid and diploid embryos reveals early zygotic transcription during cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development

Expression profile of certain mRNAs and miRNA of each class in the early embryogenesis of <i>Apis mellifera</i>.

<p>(A) Profile of mRNAs belonging to the three different classes of genes: I, II, and III. Examples of mRNAs from class I, II and III are plotted in a separated graphic for the haploid and diploid embryos. (B) Profile of miRNAs belonging to class I, II, and III. Note the differential expression and arm use among the embryo types (such as ame-<i>miR-184-3p</i> and <i>5p</i> of class II and ame-<i>miR-263-5p</i> of class II in haploids but class III in diploids). The total number of identified mRNAs for each class is as follows: class I, 667 in diploid and 475 in haploid embryos; class II, 2680 in diploid and 475 in haploid embryos; class III, 263 in diploid and 147 in haploid embryos. The total number of miRNAs for each class is as follows: class I, 90 in diploid and 72 in haploid embryos; class II, 23 in diploid and 24 in haploid embryos; class III, 37 in diploid and 67 in haploid embryos. Overall, the distribution demonstrates specific gene expression dynamics for mRNAs and miRNA in both types of embryos. The Expression and DEGseq output of the mRNAs and miRNAs plotted here are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146447#pone.0146447.s005" target="_blank">S5 Table</a>.</p

Predicted miRNA-target interactions for the diploid (A and B) and haploid (C and D) embryos of <i>Apis mellifera</i> during the early steps of embryogenesis.

<p>(A and C) Complete network and (B and D) reduced networks showing the predicted miRNA-target interactions and focusing on specific genes. Different colors and shapes identify class I, class II and class III miRNAs and genes. Note that the networks denote specific interactions for haploid and diploid embryos.</p