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This paper presents an investigation of voting behavior and whether sequential secession occurs through democratic procedures, or not. Characteristics of our model that are not found in earlier studies are the sequential timing of votes and the introduction of income differences among regions. The main results of this study demonstrate that a domino effect induces sequential secession when neighboring countries become independent. Furthermore, we analyze two model expansions: an approval vote for secession referendums by regions and a redistribution policy. Results show that voters often change their votes between secession referendums and the approval vote, that redistribution of income decreases secession, and that it is more effective when income differences are larger.

Mosquito surveillance is a fundamental component of planning and evaluating vector control programmes. However, logistical and cost barriers can hinder the implementation of surveillance, particularly in vector-borne disease-endemic areas and in outbreak scenarios in remote areas where the need is often most urgent. The increasing availability and reduced cost of 3D printing technology offers an innovative approach to overcoming these challenges. In this study, we assessed the field performance of a novel, lightweight 3D-printed mosquito light trap baited with carbon dioxide (CO2) in comparison with two gold-standard traps, the Centers for Disease Control and Prevention (CDC) light trap baited with CO2, and the BG Sentinel 2 trap with BG-Lure and CO2. Traps were run for 12 nights in a Latin square design at Rainham Marshes, Essex, UK in September 2018. The 3D-printed trap showed equivalent catch rates to the two commercially available traps. The 3D-printed trap designs are distributed free of charge in this article with the aim of assisting entomological field studies across the world., Scientific Reports, 9(1), art.no.11412; 2019

The growth and the survival of the human malaria parasite Plasmodium falciparum are critically dependent on the functions of the two organelles - the mitochondrion and the apicoplast. However, these two organelles have been known to be difficult to separate from each other when they are released from Plasmodium cell. We have been searching for the conditions with which separation of the mitochondrion and the apicoplast is achieved. In this study, we investigated how the two organelle's separation is affected when the pressure of the nitrogen gas to disrupt the Plasmodium cells by nitrogen cavitation method is lowered from the pressure regularly applied (1200 psi). The parasite cell was sufficiently disrupted even when nitrogen cavitation was carried out at 300 psi. The obtained mitochondrial sample was much less contaminated by DNA compared with the sample prepared using the gas at the regular pressure. After the fractionation by Percoll density gradient, the mitochondrion and the apicoplast from the 300 psi cell lysate exhibited different separation profiles. This is the first experimental evidence that indicates the mitochondrion and the apicoplast of P. falciparum are separable from each other.

In recent years, quite a few sub-national regions such as Scotland (UK ) and Catalonia (Spain ) have held referendums seeking independence from their respective nations, and/or are intend to do so in the future. The Scottish referendum took place in September 2014, and was agreed to in advance by both the Scottish and UK governments . The Scottish referendum was thus democratic. Furthermore, the UK government held a referendum on independence from the EU on June 2016. As a result, the UK government has decided to secede from the EU . Is this decision efficient for the British nation? What would be the case if the EU government had the right to forbid the referendum for independence?

In this chapter, we introduce a three-region model for integration and secession with policy preference and policy conflict cost. Under alternative assumptions of policy commitment of third region and integrating two regions, we show how the difference in policy preferences in two regions and the policy conflict cost influences the possibility of secession. Furthermore, we show how the policy preference in the integrated minority region affects the third region’s utility. We assume that the integration agreement works as a commitment device; therefore, integrated regions play as the first mover, and the independent region reacts to the policy agreement of the integrated regions. We consider that the policy agreement is not altered in the future because it is international treaty and announced to the public.

Facilities such as power plants, waste disposal areas, airports, prisons, and military bases are necessary and useful for society, but they can be a nuisance and noxious for neighbors, causing conflicts between governments and residents. These are therefore often called “not in my backyard” (NIMBY) facilities. Governments must construct NIMBY facilities, but their location presents many difficulties.

Haem biosynthesis appeared to be a target of malaria therapy because 5-aminolevulinic acid (ALA), a haem biosynthesis starting material, with light exposure or a high amount of ALA alone reduced Plasmodium falciparum growth to undetectable level. However, the administration of a high dose of ALA is unrealistic for clinical therapy. We found that Fe(2+) enhanced P. falciparum-killing potency of ALA and significantly inhibited the parasite growth. The intermediates of haem biosynthesis localized to the parasite organelles, and coproporphyrin III was the most accumulated intermediate. These novel findings may lead to development of a new anti-malarial drug using ALA and Fe(2+).

Protistan species belonging to the phylum Apicomplexa have a non-photosynthetic secondary plastid-the apicoplast. Although its tiny genome and even the entire nuclear genome has been sequenced for several organisms bearing the organelle, the reason for its existence remains largely obscure. Some of the functions of the apicoplast, including housekeeping ones, are significantly different from those of other plastids, possibly due to the organelle's unique symbiotic origin.

The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU’s physiological function. PfHU, which is targeted exclusively to the parasite’s plastid, bound its natural target – the plastid DNA – sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite’s survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.

Organellar DNA in mitochondria and plastids are organized with proteins into a compact structure known as the nucleoid. As the nucleoid is supposed to be the unit of inheritance for the organellar genome, it is important to understand its cytological behavior. Like plants, malaria parasites carry two organelles, the mitochondrion and the apicoplast–a non-photosynthetic plastid. However, probably because of the small size of the genome in each, visualizing the nucleoid in the Plasmodium organelles by regularly-used fluorescent dye such as DAPI, has been difficult. Here, we developed new, effective methods to visualize the organellar nucleoid in the human malaria parasite Plasmodium falciparum. With our methods using SYBR Green I or PicoGreen, nucleoids were observed in ring-stage parasites. Analyzing transfectant parasites carrying a DsRed-labelled organelle, we concluded that the parasite's mitochondrion has 1 nucleoid which is visualized with our methods. The parasite has a second nucleoid in the apicoplast, but higher concentration of the dye was required to visualize it. Our new methods would be useful for further cytological analysis of the nucleoids in the mitochondrion and the apicoplast of the malaria parasite.

We report the genome sequence of Theileria parva , an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum , and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.

Summary All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum . We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway d-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg 2+ (and enhanced by Mn 2+ ) but not by Zn 2+ . A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

A gene encoding the heme biosynthetic enzyme ferrochelatase (FC) was found in the genomic DNA databases of Plasmodium spp. The predicted amino acid sequence of malarial FC is highly conserved and fairly well conserved by comparison with other orthologues. The FC genes of P. falciparum and P. yoelii are transcribed and the mRNAs are processed to encode polypeptides of the expected amino acid sequence. The cloned cDNA for the FC of P. falciparum successfully rescued a FC-null mutant of Escherichia coli, indicating that it encodes an active enzyme. Unlike eukaryotic FCs, the malarial enzyme lacks a characteristic extension at the C-terminus. In addition, the sequence of the malarial FC resembles proteobacterial orthologues rather than eukaryotic enzymes. Strikingly, the malarial FC lacks a bipartite presequence at its N-terminus, unlike delta-aminolevulinic acid dehydratase of the same organism. This suggests an unusual intracellular distribution of heme biosynthetic enzymes, involving multiple subcellular compartments.

The enzyme delta-aminolevulinic acid dehydratase (ALAD) catalyses the second reaction in the heme biosynthetic pathway. It has been suggested previously that the malaria parasite Plasmodium falciparum imports this enzyme from the host cell for de novo heme biosynthesis. However, the parasite's genome encodes an orthologue for ALAD. Here we report molecular cloning of a complete cDNA for the parasite's intrinsic ALAD and show it rescues an ALAD-null mutant of Escherichia coli, indicating that the malarial gene encodes a functional ALAD. The malarial ALAD has a long bipartite extension at its N-terminus, which may function as a plastid targeting signal. The amino acid sequence of the enzyme is related most closely to those of plant/algal chloroplast ALADs, though the malarial version may lack the allosteric Mg2+-binding site, which is conserved among chloroplast ALADs.

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.

The DNA fragment encoding meta-cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp. strain CA10. DNA sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively. The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site. On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives. The putative polypeptide encoded by ORF3 was homologous with meta-cleavage compound hydrolases in other bacteria, and ORF3 was designated carC. The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic acid, the meta-cleavage compound of 3-methylcatechol. Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation pathway. These results suggest the possibility that CarC is a novel type of hydrolase.

Members of the genus Buchnera are intracellular symbionts harbored by the aphid bacteriocyte which selectively synthesize symbionin, a homolog of the Escherichia coli GroEL protein, in vivo. Symbionin and SymS, a GroES homolog, are encoded in the symSL operon. Northern blotting and primer extension analyses revealed that the symSL operon invariably gives rise to a bicistronic mRNA under the control of a heat shock promoter, though the amount of the symSL mRNA in the isolated symbiont did not increase in response to heat shock. The sigma32 protein that recognizes the heat shock promoter in E. coli was scarcely detected in Buchnera cells even after heat shock. Although the functionally essential regions of the Buchnera sigma32 protein were well conserved, the Buchnera rpoH gene did not complement an E. coli delta rpoH mutant. On the one hand, the A-T evolutionary pressure imposed on the Buchnera genome may have not only decreased the activity of its sigma32 but also ruined the nucleotide sequences necessary for the expression of rpoH; on the other hand, it may have facilitated expression of the symSL operon without activation by sigma32.

Induction of the enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in a medium containing straight chain saturated fatty acids was studied. The acyl-CoA dehydrogenase (ACDH) activity was induced during the exponential phase in cells grown in palmitic acid-supplemented medium, reached a maximum at the early stationary phase, and then gradually decreased thereafter. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, both existing on the multienzyme complex (HDT) involved in fatty acid beta-oxidation, were similar to that in ACDH activity. Straight chain saturated fatty acids having more than 6 carbon atoms could induce both the ACDH and HDT activities, and C13-C15 fatty acids caused the greatest induction of both activities. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase correlated with that in the amount of the alpha-subunit of HDT during the entire culture period in the medium containing palmitic acid. Surprisingly, the stoichiometry of the alpha- and beta-subunit proteins of HDT was not maintained into the stationary phase culture, though the genes encoding the alpha- and beta-subunits are tandemly coded in bacterial genomic DNA.

Three enzyme activities involved in fatty acid beta-oxidation, i.e., those of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, are exhibited by one multienzyme complex (HDT) composed of two molecules each of two peptides in Pseudomonas fragi. Using specific antisera against the two subunits of HDT, we isolated the genes encoding the subunits of HDT and designated them "faoA" (for the alpha-subunit) and "faoB" (for the beta-subunit). Their complete nucleotide sequences were determined and it was revealed that faoA and faoB, both with individual putative S.D. sequences at suitable positions, formed a cluster, in that order. The amino acid sequences deduced from the nucleotide sequences of the two genes indicated that the alpha-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the beta-subunit, encoded by faoB, consists of 390 amino acid residues lacking the first methionine of the primary product encoded by faoB. Immunoblotting of cell lysates prepared from Escherichia coli transformants carrying plasmids which possess the faoA and/or faoB gene with antisera against the subunits of HDT showed that both the faoA and faoB genes were transcribed and translated in E. coli. The overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were increased in the E. coli cells transformed with the plasmid possessing the faoA gene, suggesting that both the hydratase and dehydrogenase activities may be exhibited by the alpha-subunit of HDT.(ABSTRACT TRUNCATED AT 250 WORDS)

The mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp. is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques--Percoll density gradient centrifugation and fluorescence-activated organelle sorting--for improving the purity of mitochondria isolated from the crude organelle preparation of Plasmodium falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.

Determined efforts are being made to explore the non-photosynthetic plastid organelle of Plasmodium falciparum as a target for drug development. Certain antibiotics that block organellar protein synthesis are already in clinical use as antimalarials. However, all the indications are that these should be used only in combination with conventional antimalarials. The use of antibiotics such as doxycycline and clindamycin may reduce the development of drug resistant parasites and such means to avoid drug resistance should be explored hand-in-hand with drug development. Genomic information predicts that fatty acid type II (FAS II) and isoprenoid biosynthetic pathways are localized to the plastid. However, clinical trials with fosmidomycin (a specific inhibitor of DOXP reductase in the non-mevalonate pathway for isoprenoids) suggest it too should only be used in drug combinations. Prospects for more potent antimalarial compounds have emerged from studies of several of the enzymes involved in the FAS II pathway. Lead antibiotics such as thiolactomycin (an inhibitor of β-ketoacyl- ACP synthase) and triclosan (a specific inhibitor of enoyl-ACP reductase) have led to structurally similar, active compounds that rapidly kill ring- and trophozoite-stage parasites. The FAS II pathway is of particular interest to the pharma-industry.

Protein maturation in eukaryotic organelles requires the type I chaperonin system; this comprises chaperonin 60 (Cpn60) and its cochaperonin. We have re-examined and revised the sequence of the nuclear genes specifying organellar cochaperonins in Plasmodium falciparum (Pf). One gene encodes a typical cochaperonin (PfCpn10) whereas the other (encoding PfCpn20) specifies two Cpn10 domains arranged in tandem as in plant chloroplasts. Transfection experiments using fluorescent reporters showed specific localization of PfCpn10 to the mitochondrion and PfCpn20 to the plastid. As P. falciparum also has two Cpn60s, one of which is targeted specifically to the mitochondrion and the other exclusively to the plastid, each organelle has a distinct type I chaperonin system. Comparative sequence analysis extended these findings to several other apicomplexan parasites that have both a mitochondrion and a plastid. Phylogenetic analysis suggests the Cpn10s and Cpn20s of apicomplexans are independently monophyletic. The apicomplexan Cpn10 is phylogenetically related to other mitochondrial versions but a significant relationship between apicomplexan Cpn20s and other cochaperonins was not established.

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

Both the chromosomal and extrachromosomal components of the apicomplexan genome have been supplemented by genes from a plastid-bearing endocytobiont: probably an algal cell. The sequence of the apicomplexan plastid's vestigial genome indicates that a large number (>100) of genes of endocytobiotic origin must have transferred laterally to the host cell nucleus where they control maintenance of the plastid organelle and supply its functional components by means of post-translational protein trafficking. Should the nuclear genes prove to be less divergent phylogenetically than those left on the plastid genome, they might give better clues than we have at present to the origin of the plastid-bearing endocytobiont. Most of these nuclear genes still await discovery, but the on-going genome sequencing project will reveal the function of the organelle, as well as many “housekeeping” processes of interest on a wider front. The plastid's own protein synthetic machinery, being cyanobacterial in origin, offers conventional targets for antibiotic intervention, and this is discussed here using a structural model of elongation factor Tu. Uncovering the vital function(s) of the plastid organelle will provide new drug targets.

Buchnera sp., an intracellular symbiont of the pea aphid (Acyrthosiphon pisum Harris), is a close phylogenetical relative of Escherichia coli, and synthesizes a large amount of symbionin, a GroEL homolog. The other heat shock protein homologs, which are not expressed as much as symbionin, have not been studied yet. In this study, we cloned the dnaK and dnaJ genes of Buchnera, and revealed that its DnaK and DnaJ are structurally very similar to those of E. coli. Amino acid residues and motifs proposed so far to be essential for the function of the E. coli DnaK and DnaJ were completely conserved in the Buchnera counterparts. However, Buchnera dnaKJ operon could not fully complement mutations of either dnaK or dnaJ of E. coli. This is probably because of a difference in net charge of DnaK and DnaJ between Buchnera and E. coli, and a unique structure of Buchnera DnaJ that prevents heterologous components from operating in concert. Buchnera dnaK and dnaJ formed an operon whose transcription is governed by a promoter structurally homologous to heat shock promoters of E. coli, although the cellular amount of dnaKJ mRNA was not affected by heat shock. Two inverted repeats flanking both sides of E. coli dnaJ were also found in the gene of Buchnera at the corresponding positions, suggesting that expression ratio of DnaK to DnaJ is regulated in a similar manner in the two organisms.

Leucocytozoon caulleryi, a haemosporidian parasite of the chicken (Gallus gallus domesticus), can be highly pathogenic and often fatal. Although this parasite is extremely relevant to veterinary science, knowledge of its genomic features is limited. To gain information applicable to developing novel control methods for the parasite, we analyzed the apicoplast genome of L. caulleryi. This extranuclear organellar DNA of 85.1 % A + T and a unit of 34,779 bp was found to encode almost the same set of genes as the plastid genome of Plasmodium falciparum, including 16 tRNA and 30 protein coding genes, and except for one open reading frame, ORF91 absent in L. caulleryi. As in P. falciparum, the L. caulleryi apicoplast DNA contains two sets of a unique inverted repeat (IR), each one 5,253 bp and encoding genes specifying one large and one small rRNA subunit and nine tRNAs but no protein, and separated by a unique 13 bp sequence. Studies of several haemosporidian apicoplast DNA sequences have identified a corresponding IR region; however, none of these studies has looked at the complete sequence, even for well-studied species such as P. falciparum. Phylogenetic studies using a concatenated amino acid sequence based on the open reading frames confirmed the close relationship between L. caulleryi and Plasmodium spp. In this study, we determined the nucleotide sequence of the entire L. caulleryi apicoplast genome, including the region connecting the two IR units. This is the first report of the complete nucleotide sequence of a haemosporidian apicoplast DNA with a canonical IR.

Nucleotide sequences encoding GroEL homologs of intracellular symbionts in three closely interrelated aphids were compared with one another and that for GroEL. It was suggested that in these proteins a particular position is highly susceptible to amino acid substitution, through which the GroEL homologs of symbionts seemed to have acquired a unique function on top of the activity as molecular chaperone. This may represent a rare example of non-neutral evolution of molecule under the positive selection pressure.