Browsing by Author "Mireji, Paul O"

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Antimutagenic effect of Kenyan Tea cultivars in a bacterial test system
(2014) Mbuthia, Karori; Wachira, Francis; Ngure, Raphael; Mireji, Paul O; Wachira, Sabina
The antimutagenic effects of the aqueous tea extracts from Kenyan black, green and purple cultivars were evaluated by the Ames te st using Salmonella typhimurium tester strains TA 1538. Results obtained showed that tea had no toxicity or mutagenic activity at a concentration of 20% (w/v) unlike the mut agen sodium azide. However, using the formulae, percentage inhibition = [1 - T/M] ×10 0 where T is number of revertants per plate in presence of mutagen and test sample and M is number of revertants per plate in positive control, tea extracts had a significant (P<0.05) antimutagenic activity where the percent inhibition was 65% for green te a, 38% for purple tea and 19.17% for black tea. This was attributed to the radical scavenging activity of polyphenols. There is need therefore to carry out further research to help understand the precise mechanism of action especially for black and purple teas, and to explore other beneficial effects that these polyphenols may have, before they can be adopted for therapeutic use.
Aquaporins Are Critical for Provision of Water during Lactation and Intrauterine Progeny Hydration to Maintain Tsetse Fly Reproductive Success
(2014) Benoit, Joshua B; Hansen, Immo A; Attardo, Geoffrey M; Michalková, Veronika; Mireji, Paul O; Bargul, Joel L; Drake, Lisa L; Masiga, Daniel K; Aksoy, Serap
Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4–6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20–25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.
Blood Meal Analysis and Virus Detection in Blood-Fed Mosquitoes Collected During the 2006–2007 Rift Valley Fever Outbreak in Kenya
(2014) Lutomiah, Joel; Omond, David; Masiga, Daniel; Mutai, Collins; Mireji, Paul O; Ongus, Juliette; Linthicum, Ken J; Sang, Rosemary
Background: Rift Valley fever (RVF) is a zoonosis of domestic ruminants in Africa. Blood-fed mosquitoes collected during the 2006–2007 RVF outbreak in Kenya were analyzed to determine the virus infection status and animal source of the blood meals. Materials and Methods: Blood meals from individual mosquito abdomens were screened for viruses using Vero cells and RT-PCR. DNA was also extracted and the cytochrome c oxidase 1 (CO1) and cytochrome b (cytb) genes amplified by PCR. Purified amplicons were sequenced and queried in GenBank and Barcode of Life Database (BOLD) to identify the putative blood meal sources. Results: The predominant species in Garissa were Aedes ochraceus, (n=561, 76%) and Ae. mcintoshi, (n=176, 24%), and Mansonia uniformis, (n=24, 72.7%) in Baringo. Ae. ochraceus fed on goats (37.6%), cattle (16.4%), donkeys (10.7%), sheep (5.9%), and humans (5.3%). Ae. mcintoshi fed on the same animals in almost equal proportions. RVFV was isolated from Ae. ochraceus that had fed on sheep (4), goats (3), human (1), cattle (1), and unidentified host (1), with infection and dissemination rates of 1.8% (10/561) and 50% (5/10), respectively, and 0.56% (1/176) and 100% (1/1) in Ae. mcintoshi. In Baringo, Ma. uniformis fed on sheep (38%), frogs (13%), duikers (8%), cattle (4%), goats (4%), and unidentified hosts (29%), with infection and dissemination rates of 25% (6/24) and 83.3% (5/6), respectively. Ndumu virus (NDUV) was also isolated from Ae. ochraceus with infection and dissemination rates of 2.3% (13/561) and 76.9% (10/13), and Ae. mcintoshi, 2.8% (5/176) and 80% (4/5), respectively. Ten of the infected Ae. ochraceus had fed on goats, sheep (1), and unidentified hosts (2), and Ae. mcintoshi on goats (3), camel (1), and donkey (1). Conclusion: This study has demonstrated that RVFV and NDUV were concurrently circulating during the outbreak, and sheep and goats were the main amplifiers of these viruses respectively.
Changes in genotypes of Plasmodium falciparum human malaria parasite following withdrawal of chloroquine in Tiwi, Kenya
(Elsevier, 2012) Mang’era, Clarence M.; Mbai, Fiona N; Omedo, Irene A; Mireji, Paul O; Omar, Sabah A
Chloroquine (CQ) drug was withdrawn in 1998 as a first-line treatment of uncomplicated malaria in Kenya. This was in response to resistance to the drug in Plasmodium falciparum malaria parasite. Investigations were conducted to determine prevalence of CQ resistance genotypes in the parasites in Tiwi, a malaria endemic town in Kenya, before and about a decade after the withdrawal of the drug. Blood samples were collected and spotted on filter papers in 1999 and 2008 from 75 and 77 out-patients respectively with uncomplicated malaria. The sampling was conducted using finger pricking technique. DNA was extracted from individual spots in the papers and screened for the presence of P. falciparum chloroquine resistance transporter (Pfcrt) and multi drug resistance (Pfmdr-1) markers using nested PCR. Nature of mutations (haplotypes) in the Pfcrt and Pfmdr-1 markers in the samples were confirmed using dot blot hybridization technique. Changes in pattern of CQ resistance in the parasite samples in 1999 and 2008 were assessed by Chi Square test. There was a significant (P < 0.05) reduction in CQ resistant genotypes of the parasite between 1999 and 2008. Pfmdr and Pfcrt CQ resistant genotypes in 2008 reduced to 54.10 and 63.64% respectively, from 75.39 and 88.0% respectively in 1999. This reduction was accompanied by emergence of Pfcrt specific CQ sensitive (IEK) and intermediate/partially CQ resistant (MET) haplotypes. Results suggest significant reversal of the phenotype of the parasite from chloroquine resistant to wild/sensitive type. The novel haplotypes indicates transitional phase of the parasite to the wild type. Current prevalence of chloroquine resistant genotype is definitely above the threshold for efficacious re-introduction of chloroquine for treatment of malaria in Tiwi.
Chemosensory receptors in tsetse flies provide link between chemical and behavioural ecology
(2014) Masiga, Daniel; Obiero, George; Macharia, Rosaline; Mireji, Paul O; Christoffels, Alan
Tsetse flies survive in a variety of environments across tropical Africa, often rising to large numbers, despite their low birth rate of one offspring every seven to nine days. They use olfactory receptors to process chemical signals in their environments to find food, escape from predators, and locate suitable larviposition sites. We discuss the identification of odorant and gustatory receptors in Glossina morsitans morsitans and the role genomics could play in management of nuisance insects
Deciphering the reproductive protein-protein interaction network in Anopheles gambiae with Drosophila melanogaster as a framework
(2011) Achinko, Daniel; Mireji, Paul O; Catteruccia, Flaminia; Masiga, Dan
Protein-protein interactions (PPIs) are the most fundamental biological processes at the molecular level. The experimental methods for testing PPIs are time-consuming and are limited by analogs for many reactions. As a result, a computational model is necessary to predict PPIs and to explore the consequences of signal alterations in biological pathways. Reproductive control of the vector Anopheles gambiae using transgenic techniques poses a serious challenge. To meet this challenge, it would help to define the biological network involving the male accessory gland (MAG) proteins responsible for successful formation of the mating plug [1]. This plug forms in the male and is transferred to the female during mating, hence initiating the PPIs in both sexes. As is the case in Drosophila melanogaster, a close relative of A. gambiae, some MAG proteins responsible for the formation of the mating plug have been shown to alter the post-mating behavior of females.
Differential induction of proteins in Anopheles gambiae sensu stricto (Diptera: Culicidae) larvae in response to heavy metal selection
(2006) Mireji, Paul O; Keating, Joseph; Kenya, Eucharia; Mbogo, Charles; Nyambaka, Hudson; Osir, Ellie; Githure, John; Beier, John
Investigations were conducted to establish the magnitude and pattern of differential expression of proteins due to generational selection of third instar Anopheles gambiae s.s. Giles larvae by cadmium, copper and lead heavy metals, the three possible common urban pollutants. A susceptible strain of A. gambiae s.s. third instar larvae was separately placed under selection pressure with cadmium, copper and lead at LC30 and controls through five generations. First, third and fifth generation selection survivors were screened for differentially expressed proteins relative to non-exposed control by two-dimensional gel electrophoresis. Distribution patterns of the spots were analyzed by χ2 or Fishers' exact test and variations in expressions between and within generations by ANOVA. Most differentially expressed spots were acidic and of low molecular weight among all metals and generations. Type of heavy metal and generation were the main indicators of variations in differential expressions. Variation between generations was most significant among cadmium-selected populations of which the most number of spots were induced in the fifth generation. Most spots were induced in the copper-selected population in the third generation. The induced protein spots may be the products from respective genes that respond to heavy metals and counter their toxicity, thus building A. gambiae s.s. tolerance to these pollutants. The differential pattern and magnitude of expressed spots have potential application as molecular markers for assessment of anopheline adaptation status to heavy metals, and provide insight into the extent of environmental pollution
Expression of metallothionein and α-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae)
(2010) Mireji, Paul O; Keating, Joseph; Hassanali, Ahmed; Impoinvil, Daniel E; Mbogo, Charles M; Muturi, Martha N; Nyambaka, Hudson; Kenya, Eucharia U; Githure, John I; Beier, John C
Anopheles mosquitoes have been shown to adapt to heavy metals in their natural habitats. In this study we explored the possibility of using Anopheles gambiae sensu stricto as bio-reporters for environmental heavy metal pollution through expressions of their metal-responsive metallothionein and α-tubulin genes. The study was undertaken with third instar larvae after selection by cadmium, copper, or lead at LC30 through five successive generations. Expression levels were determined in the 5th generation by semi-quantitative RT-PCR on the experimental and control populations. The data were analyzed using one-way ANOVA. The highest metallothionein (F3, 11=4.574, P=0.038) and α-tubulin (F3, 11=12.961, P=0.002) responses were observed in cadmium-tolerant treatments. There was significantly higher expression of metallothionein in cadmium or copper treatments relative to the control (P=0.012), and in cadmium than in lead treatments (P=0.044). Expressions of α-tubulin were significantly higher in cadmium than in control treatments (P=0.008). These results demonstrate the capacity of An. gambiae s.s. to develop tolerance to increased levels of heavy metal challenge. The results also confirm the potential of heavy metal-responsive genes in mosquitoes as possible bio-indicators of heavy metal environmental pollution. How the tolerance and expressions relate to An. gambiae s.s. fitness and vectorial capacity in the environment remains to be elucidated.
Juvenile hormone and insulin suppress lipolysis between periods of lactation during tsetse fly pregnancy
(Elsevier, 2013) Baumann, Aaron A; Benoit, Joshua B; Michalkova, Veronika; Mireji, Paul O; Attardo, Geoffrey M; Moulton, John K; Wilson, Thomas G; Aksoy, Serap
Tsetse flies are viviparous insects that nurture a single intrauterine progeny per gonotrophic cycle. The developing larva is nourished by the lipid-rich, milk-like secretions from a modified female accessory gland (milk gland). An essential feature of the lactation process involves lipid mobilization for incorporation into the milk. In this study, we examined roles for juvenile hormone (JH) and insulin/IGF-like (IIS) signaling pathways during tsetse pregnancy. In particular, we examined the roles for these pathways in regulating lipid homeostasis during transitions between non-lactating (dry) and lactating periods. The dry period occurs over the course of oogenesis and embryogenesis, while the lactation period spans intrauterine larvigenesis. Genes involved in the JH and IIS pathways were upregulated during dry periods, correlating with lipid accumulation between bouts of lactation. RNAi suppression of Forkhead Box Sub Group O (FOXO) expression impaired lipolysis during tsetse lactation and reduced fecundity. Similar reduction of the JH receptor Methoprene tolerant (Met), but not its paralog germ cell expressed (gce), reduced lipid accumulation during dry periods, indicating functional divergence between Met and gce during tsetse reproduction. Reduced lipid levels following Met knockdown led to impaired fecundity due to inadequate fat reserves at the initiation of milk production. Both the application of the JH analog (JHA) methoprene and injection of insulin into lactating females increased stored lipids by suppressing lipolysis and reduced transcripts of lactation-specific genes, leading to elevated rates of larval abortion. To our knowledge, this study is the first to address the molecular physiology of JH and IIS in a viviparous insect, and specifically to provide a role for JH signaling through Met in the regulation of lipid metabolism during insect lactation.
Larvicidal Activity of Selected Aloe Species Against Aedes aegypti (Diptera: Culiciade)
(2014) Chore, Judith K; Obonyo, Meshack; Wachira, Francis N; Mireji, Paul O
Management of mosquito vectors by current classes of mosquitocides is relatively ineffective and necessitates prospecting for novel insecticides with different modes of action. Larvicidal activities of 15 crude extracts from three geographically isolated Aloe ngongensis (Christian), Aloe turkanensis (Christian), and Aloe fibrosa (Lavranos & L.E.Newton) (Xanthorrhoeaceae) species (five each) were evaluated against Aedes aegypti (Linnaeus in Hasselquist) (Diptera: Culiciade L.) yellow fever mosquito. Freshly collected leaves were separately shade-dried to constant weight at room temperature (25 ± 2°C) and powdered. Each powder was macerated in solvents of increasing polarity (hexane, chloroform, ethyl acetate, acetone, and methanol) for 72 h and subsequently filtered. Third-instar larvae (n = 25) of the mosquito were exposed to the extracts at different concentrations for 24 h to establish dose response relationships. All the fractions of A. ngongensis were active below 1 mg/ml except A. fibrosa and A. turkanensis. The highest activity (LC50) mg/ml was obtained with extracts of A. fibrosa hexane (0.05 [0.04–0.06]), followed by A. ngongensis hexane (0.11 [0.08–0.15]) and A. turkanensis ethyl acetate (0.11 [0.09–0.12]). The activities are apparently Aloe species specific and extraction solvent dependent. These findings suggest that extracts from selected Aloe species have mosquitocidal principles that can be exploited in development of new insecticides.
Life stage and tissue speciation of cathepsin B (AGAP004533) derives different functional properties in the G3 strain of the mosquito Anopheles gambiae
(2012) Achinko, Daniel; Masiga, Dan; Mireji, Paul O; Catteruccia, Flaminia
Cathepsin B is a lysosomal papain-like cysteine peptidase that is expressed in all tissues and functions primarily as an exopeptidase through its carboxydipeptidyl activity. Together with other cathepsins, it is involved in the degradation of proteins, proenzyme activation, antigen processing, metabolism and apoptosis. AGAP004533 is a cathepsin B peptidase of 337 amino acids known to be found on the mating plug. This plug is known to be produced in the male Anopheles gambiae mosquito and transferred to the female during mating [1]. The female digests this plug in 24 h. The protein is expressed in all life stages of the mosquito and in all tissues of the adult. We cloned and sequenced the protein in the larvae and pupae stages and all reproductive tissues (spermatheca, atria and ovary of the female; testes and male accessory glands (MAGs) of the male) of the G3 mosquito strain. These sequences were analysed with Geneious 5.5.5 and cLc workbench 6.6.1 software. Within the coding sequence, two single mutations at C584T (juvenile stages) and nucleotide A14T (ovary) were identified. The latter translates into a glutamine for leucine (Q6L), which causes the loss of the signal peptide due to loss of five amino acids at the N-terminal region of the protein sequence, meanwhile the former translates into an alanine for valine (A195V). Both mutations cause structural modifications within the secondary structure of the protein that eventually affect its 3D conformation. The sequences in the artria and spermatheca showed insertion of a cytosine at nucleotide 1010, which translates to a proline for a leucine (P337L) substitution, and hence loss of the stop codon at amino acid 338. This loss causes an extension of 14 amino acids at the carboxylic end of the protein, resulting in secondary structure modification. The sequence for the testes appeared transposed, and hence was not considered in the analysis. All the sequences translated on the same frame except for that of the ovary, which translated on a different frame. Protein BLAST of these sequences at NCBI using the blosum62 matrix, identified with AGAP004533 of Anopheles gambiae alongside other mosquito species, although that of the artria and spermatheca also identified with species of distant taxa such as Manduca sexta (FM957999.1) and Gallus gallus (NM205371.1). This relation was due to the amino acid extensions at the carboxylic end relating to parasite killing in the former, and embryonic apoptosis in the latter. Transcription factor predictions on all sequences identified equal binding sites (T00821, T00752), and that of the male accessory glands identified an extra binding site (T00360) known in humans as a bifunctional protein nuclear cytoplasmic O-N-acetylglucosaminidase and acetyltransferase. This site also has alternative splicing functions, which could be important for the variations observed in this gene. Sequence variations of this protein in the different stages and tissues of the mosquitoes may also be highly related to their functions and relative positions in the same or different biological processes within the various tissues. In-depth analysis of the reproductive role of AGAP004533 will help in reproductive control of the vector
Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans
(2014) Obiero, George FO; Mireji, Paul O; Nyanjom, Steven RG; Christoffels, Alan; Robertson, Hugh M; Masiga, Daniel K
Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO2 were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.
RESEARCH Open Access High-resolution melting analysis reveals low Plasmodium parasitaemia infections among microscopically negative febrile patients in western Kenya
(2014) Kipanga, Purity N; Omondi, David; Mireji, Paul O; Sawa, Patrick; Masiga, Daniel K; Villinger, Jandouwe
Background: Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting. Methods: Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections. Results: The coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials. Conclusions: The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions
RESEARCH Open Access Sex-specific induction of CYP6 cytochrome P450 genes in cadmium and lead tolerant Anopheles gambiae
(2013) Musasia, Fauzia K; Isaac, Alfred O; Masiga, Daniel K; Omedo, Irene A; Ochieng, Richard; Mireji, Paul O; Mwakubambanya, Ramadhan
Background: Anopheles gambiae, one of the main Afro-tropical mosquito vector of malaria, has adapted to heavy metals in its natural habitat, and developed resistance to most conventional insecticides. Investigations were conducted to establish an association between tolerance to cadmium or lead-heavy metals, and expression of specific genes for cytochrome p450 enzymes associated with pyrethroid resistance in the mosquito. Methods: Juvenile aquatic stages of the mosquito were selected for tolerance to cadmiun or lead through chronic exposure of the stages to maximum acceptable toxicant concentrations (MATCs) of the metals. Using real-time quantitative polymerase chain reaction (qPCR), three replicates each of male or female cadmium or lead-tolerant individuals and relevant controls were separately screened for expression of CYP6M2 , CYP6P3 and CYP6Z1 genes. The variance in expression levels of the genes amongst the treatments was compared by ANOVA statistical tool. Results: Expressions of all the genes were significantly lower (P <0.05) in females than in males. Within gender, there 1.3 - 2.3 or 3.1-4.2-fold reduction in expression of the genes in cadmium or lead selected than respective control populations. Expression of all the classes of gene was elevated in cadmium selected female populations relative to their respective controls. Conclusion: These findings suggest that tolerance to cadmium or lead in the mosquito can influence response in cytochrome p450 genes associated with metabolism of pyrethroids in the mosquito in a sex-specific manner. This can, in turn, affect sensitivity of the mosquito to pyrethroids and other xenobiotics associated with these genes, with potential implications in mosquito vector control operations.