Triatominae–Trypanosoma cruzi/T. rangeli: Vector–parasite interactions

Of the currently known 140 species in the family Reduviidae, subfamily Triatominae, those which are most important as vectors of the aetiologic agent of Chagas disease, Trypanosoma cruzi, belong to the tribes Triatomini and Rhodniini. The latter not only transmit T. cruzi but also Trypanosoma rangeli, which is considered apathogenic for the mammalian host but can be pathogenic for the vectors. Using different molecular methods, two main lineages of T. cruzi have been classified, T. cruzi I and T. cruzi II. Within T. cruzi II, five subdivisions are recognized, T. cruzi IIa–IIe, according to the variability of the ribosomal subunits 24Sα rRNA and 18S rRNA. In T. rangeli, differences in the organization of the kinetoplast DNA separate two forms denoted T. rangeli KP1+ and KP1−, although differences in the intergenic mini-exon gene and of the small subunit rRNA (SSU rRNA) suggest four subpopulations denoted T. rangeli A, B, C and D. The interactions of these subpopulations of the trypanosomes with different species and populations of Triatominae determine the epidemiology of the human-infecting trypanosomes in Latin America. Often, specific subpopulations of the trypanosomes are transmitted by specific vectors in a particular geographic area. Studies centered on trypanosome–triatomine interaction may allow identification of co-evolutionary processes, which, in turn, could consolidate hypotheses of the evolution and the distribution of T. cruzi/T. rangeli-vectors in America, and they may help to identify the mechanisms that either facilitate or impede the transmission of the parasites in different vector species. Such mechanisms seem to involve intestinal bacteria, especially the symbionts which are needed by the triatomines to complete nymphal development and to produce eggs. Development of the symbionts is regulated by the vector. T. cruzi and T. rangeli interfere with this system and induce the production of antibacterial substances. Whereas T. cruzi is only subpathogenic for the insect host, T. rangeli strongly affects species of the genus Rhodnius and this pathogenicity seems based on a reduction of the number of symbionts.     

Trypanosoma rangeli isolates of bats from Central Brazil: Genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T. rangeli, T. dionisii, T. cruzimarinkellei and T. cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus planirostris were identified as T. rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T. rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T. rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T. rangeli and T. cruzi.     

Infestation of peridomestic Attalea phalerata palms by Rhodnius stali,a vector of Trypanosoma cruzi in the Alto Beni,Bolivia

Objectives To determine (i) whether peridomestic Attalea phalerata palms in fragmented human‐occupied areas of the Alto Beni, Bolivia, are infested by triatomines; (ii) the specific status of triatomines captured in the area; and (iii) the rate of natural Trypanosoma cruzi infection among those triatomines. Methods One hundred and twenty‐five live‐bait traps were used to sample 47 A. phalerata palms in three Alto Beni localities. Active search for vectors was also performed in 10 chicken coops and three rice storage units. Only Rhodnius specimens were found. As nymphs of closely related Rhodnius species are morphologically undistinguishable, and because of controversy in the literature regarding which Rhodnius species occur in Bolivia, collected insects were identified through molecular taxonomy. Phylogenetic analyses of DNA sequences obtained for a fragment of the mitochondrial cytochrome b gene and for the nuclear ITS‐2 ribosomal region were used as molecular markers. Natural infection rates were determined using a pair of primers that PCR‐amplify a 330‐bp fragment of the parasite’s kDNA. Results Twelve nymphs were captured in five A. phalerata palms (from two of the three localities studied), and an adult was collected from a chicken coop in Iniqua (and morphologically identified as Rhodnius stali). All nymphs (as well as the adult) were molecularly identified as R. stali based on the two molecular markers used. A single nymph was found to be infected with T. cruzi. Conclusions Attalea phalerata palms represent an important sylvatic ecotope occupied by R. stali in the Alto Beni region of Bolivia, where there are signs of T. cruzi transmission to humans, despite the preliminary indication of low level of natural infection of the vectors.     

Karyotype variability in KP1(+) and KP1(−) strains of Trypanosoma rangeli isolated in Brazil and Colombia

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(−) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T. rangeli strains into two groups coinciding with the KP1(+) and KP1(−) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(−) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1(−) strains of T. rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA.     

Trypanosoma cruzi I-III in southern Brazil causing individual and mixed infections in humans, sylvatic reservoirs and triatomines

The aim of this study was to characterise Discrete Typing Units (DTUs) of 28 isolates of Trypanosoma cruzi from humans (15), triatomines (9), and opossums (4) in the state of Paraná, southern Brazil. For this purpose, we analysed the size polymorphism at the 3′ end of the 24Sα ribosomal RNA gene (rRNA) and the restriction fragment length polymorphism (RFLP) of the partial 5′ sequence of the mitochondrial Cytochrome Oxidase subunit II gene (COII). Band patterns of the isolates were compared with reference samples of T. cruzi I (Silvio X10 and Col 17G2), T. cruzi II (Esmeraldo and JG), T. cruzi III (222 and 231), T. cruzi IV (CAN III), T. cruzi V (SO3 cl5), and T. cruzi VI (CL Brener). Our results confirmed that rRNA analysis is of limited use for assessing T. cruzi DTUs. COII RFLP analysis was suitable for screening, but for one isolate it was necessary to determine the COII partial sequence to identify the DTU. Only one of the isolates from humans belonged to T. cruzi I; 13 isolates belonged to T. cruzi II and one to T. cruzi III. The four isolates from opossums and five isolates from triatomines were identified as T. cruzi I. Four isolates from triatomines showed patterns of both T. cruzi I and II, indicating mixed infections. This study contributes to the characterisation of the dynamics of T. cruzi populations in southern Brazil.     

Heterogeneity found in the cagA gene of Helicobacter pylori from Japanese and non-Japanese isolates

We analyzed cagA genes from Helicobacter pylori strains isolated from Japanese and non-Japanese individuals for differences that could be associated with variations in virulence. The cagA genes from Japanese isolates (n = 12) and non-Japanese American Type Culture Collection (ATCC) strains (n = 4) were sequenced and compared with three published sequences. Phylogenetic analysis resolved two distinct clusters with a genetic distance of 0.1602. Similarity plot analysis of the amino acid sequences identified two highly variable regions of which each was unique to the Japanese and non-Japanese isolates, respectively. Furthermore, nucleic acid sequence analysis revealed that the multiple repeated sequences present in cagA may have been generated by homologous recombination and/or misaligned replication to promote variation in the cagA gene products. Our data indicate that alleic variations in the H. pylori genome exist between isolates from Japanese and non-Japanese subjects and that distinct H. pylori populations may be circulating in different geographical regions. Phylogenetic analysis did not reveal any association of a specific CagA type with a particular disease. Although extensive alterations were found in the cagA gene, none of the isolates contained a prematurely terminated CagA protein. The cagA gene may be advantageous to H. pylori, possibly by aiding its escape from host immune recognition by antigen modulation. Thus, this ability to elude the host immune system may contribute to an increased risk for gastric disease. Received: March 2, 2000 / Accepted: July 7, 2000     

Characterization of the metacaspase 1 gene in Plasmodium vivax field isolates from southern Iran and Italian imported cases

Plasmodium vivax is still the more prevalent human Plasmodium outside Africa and despite this fact, there is still a deep lack of knowledge on its biology. Metacaspases are cysteine proteases related to metazoan caspases, involved in programmed cell death. Here, we have characterized the P. vivax metacaspase 1 gene in a total of 63 vivax isolates, 32 isolates collected in southern Iran and 31 Italian imported isolates originating from 12 different endemic countries. We have firstly identified DNA size polymorphism in P. vivax metacaspase 1 gene. A total of four different allelic sizes were found, resulting from the insertion of 1 to 4 tandem repeat units located within the intronic region of the P. vivax metacaspase 1. Similarly, we also have identified four distinct allelic types by using vivax merozoite surface protein-1 size polymorphism analysis.     

Molecular analysis of surface glycoprotein multigene family TrGP expressed on the plasma membrane of Trypanosoma rangeli epimastigotes forms

Trypanosoma rangeli, a non-pathogenic hemoflagelate that in Central and South America infects humans, shares with Trypanosoma cruzi reservoirs and triatomine vectors, as well as geographical distribution. Recently, we have described in T. rangeli a truncated gene copy belonging to the group II of the trans-sialidase superfamily (TrGP). This superfamily, collectively known in T. cruzi as gp85/TS, includes members that are involved in host cell invasion and infectivity. To confirm the presence of this superfamily in the genome of T. rangeli and obtain a better knowledge of its characteristics, we designed a PCR and RT-PCR cloning strategy to allow sequence analysis of both genomic and transcribed copies. We identified two full-length copies of TrGP, some pseudogenes, and N- and C-terminal sequences of several genes. We also analyzed the expression and cellular localization of these proteins in epimastigote forms of a Venezuelan T. rangeli isolate using polyclonal antibodies made against a recombinant peptide from the N-terminal region of a TrGP member. We confirmed that TrGP is a multigenic family that shares many features with T. cruzi gp85/TS, including the telomeric location of some of its members, and by immunofluorescence analysis that its location is at the surface of the parasite.     

Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies

Panama disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1α and the mitochondrial small subunit ribosomal RNA genes for F. oxysporum strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of F. oxysporum. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to “F. oxysporum f. sp. cubense” with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1α and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.     

Molecular identification and phylogenetic analysis of Trypanosoma evansi from dromedary camels (Camelus dromedarius) in Egypt, a pilot study

Animal trypanosomiasis is one of the major constraints of livestock industry in developing countries. In the present study, prevalence of Trypanosome evansi was assessed in the blood of dromedary camels (Camelus dromedarius) brought to Al Bassatein abattoir, Cairo, Egypt, by mouse inoculation test out of 84 tested camels, 4 animals (4.7%) were infected. Molecular analysis was achieved by PCR amplification and sequence analysis of part of ribosomal RNA gene including 18S, ITS1, 5.8S and ITS2 regions. Despite the conserved nature of 18S region, ITS region showed obvious heterogeneity compared to analogous sequences in database. Analysis of transferrin receptor encoding gene (ESAG6) showed variable repertoire in the studied isolates, which may indicate to a novel structure of T. evansi population from Egypt and/or a difference in host range. Furthermore, analysis of variable surface glycoprotein RoTat 1.2 gene marker revealed some heterogeneity at this gene locus. To our knowledge, this is the first molecular analysis of T. evansi in Egypt.     

Detection of antibodies in sera from Chagas' disease patients using a Trypanosoma cruzi immunodominant recombinant antigen

A Trypanosoma cruzi DNA fragment encoding an immunodominant repetitive antigen (H49) was subcloned into a protein purification and expressed system. Purified H49 peptide reacted specifically in an enzyme-linked immunosorbent assay (ELISA) with sera from T. cruzi-infected patients, but not with sera from patients with other parasitic diseases such as leishmaniasis and T. rangeli-infection. The H49 recombinant ELISA was able to detect specific antibodies in 84% of chronic chagasic serum samples tested. One of the major advantage of the recombinant ELISA for serodiagnosis of chronic Chagas’disease resides in its high specificity (100%). Our data suggest that recombinant peptides could provide a practical basis for specific diagnosis tests for Chagas’disease.     

Molecular variation of Taenia solium in the world

Complete nucleotide sequences of the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit I (CO I) genes from various isolates of Taenia solium were examined. Eleven isolates were analyzed; two isolates from China, two isolates from Indonesia, one isolate each from India, Thailand, Mexico, Ecuador, Peru, Mozambique and Tanzania. In both genes, two isolates from Indonesia shared the same sequences. Similarly, the isolate from Mexico shared same sequences with that from Peru, and the isolate from Mozambique shared same sequences with that from Tanzania. Phylogenetic trees inferred from different mitochondrial genes yielded almost the same topology. Both the UPGMA and NJ-trees were also very similar. These trees indicate that T. solium may be diverged to 2 genetic groups; isolates from Asia form one group and isolates from Africa and Latin America belong to the other. It seems that T. solium prevalent in Africa and in Latin America shares the related origin and has recently been introduced to each area, perhaps with domestic pigs or human.     

Phylogeography of Androctonus species (Scorpiones: Buthidae) in Tunisia: Diagnostic characters for linking species to scorpionism

A fragment of the mitochondrial (mt) 16S ribosomal RNA gene was amplified by PCR and sequenced from individual adult scorpions of the genus Androctonus, which were sampled from central and southern Tunisia and identified using an explicit set of morphological characters. Phylogenetic analyses placed the mtDNA haplotypes in three well-supported monophyletic lineages, corresponding to the morphospecies Androctonus aeneas, Androctonus amoreuxi and Androctonus australis. The latter species was the most abundant and widespread, and it was characterized by two mtDNA sub-lineages each of which predominated only north or south of the Chott el Jerid, a seasonally flooded saline depression that divides non-Mediterranean Tunisia. The divergence of the two mtDNA lineages was dated by mtDNA molecular clocks, indicating that the formation of the Chott el Jerid is unlikely to have been the barrier generating the vicariant evolution of the two lineages of A. australis, although it may have impeded their mixing following secondary contact. Both regional mtDNA lineages were found in A. australis hector and A. australis garzonii, indicating that these two morphological forms are neither monophyletic nor geographically isolated and, therefore, should not be treated as species or subspecies. It is recommended that no subspecies of A. australis should be recognized in North Africa and toxicologists should cease the taxonomic error of referring to a species “Androctonus australis Hector”. The morphological form “hector” has no proven association with an increased risk of scorpionism compared with “garzonii”. However, it might be prudent to produce anti-venom in Tunisia by using both morphological forms of A. australis collected each side of the Chott el Jerid, because of the evidence for regional variation in toxins. The highest risk for scorpion stings occurs in the central region, where the new diagnostic markers should be used to discover any association between Androctonus species and scorpionism.     

Sequences of the second internal transcribed spacer of ribosomal DNA for three species of <Emphasis Type="Italic">Trichostrongylus</Emphasis> (Nematoda: Trichostrongylidae) from sheep in Russia

Summary For the first time, DNA sequence data were obtained for three species of Trichostrongylus from Russia. Internal transcribed spacer (ITS-2) of ribosomal DNA was sequenced for T. axei, T. colubriformis and T. probolurus from sheep from the Moscow region. ITS-2 rDNA length was estimated as 238 nucleotides for T. colubriformis and T. probolurus and 237 nucleotides for T. axei. The G+C content of the ITS-2 sequences of T. colubriformis, T. axei and T. probolurus were 31 %, 32 % and 34 % respectively. The level of interspecific differences in ITS-2 of rDNA of T. axei, T. probolurus and T. colubriformis ranged from 3 to 4 %. The ITS-2 sequences from the Russian specimens were compared with those of T. axei, T. probolurus and T. colubriformis from Australia and Germany. Intraspecific variation ranged from 0 % in T. colubriformis to 3.0 % in T. axei.     

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.     

Mesocestoides litteratus (Batsch, 1786) (Cestoda: Cyclophyllidea: Mesocestoididae) from the red fox: Morphological and 18S rDNA characterization of European isolates

Summary Mesocestoides litteratus (Batsch, 1786) (Cestoda: Cyclophyllidea: Mesocestoidae) is a common parasite of the red fox (Vulpes vulpes) and other carnivores across Europe. There has been considerable debate as to the validity of M. litteratus and other closely related, often sympatric species of Mesocestoides. We examine isolates of M. litteratus from red foxes in the Czech Republic, Slovakia and Spain both morphometrically and by characterization of 18S rDNA. Morphometric ranges of all isolates confirmed their identity as M. litteratus and were usually within the range published formerly. The sequences of 18S rDNA of one or two isolates from each country were analysed. The sequences were the same and distinct from all published Mesocestoides 18S sequences with the exception of tetrathyridia from a lizard in the Czech Republic, which was identical to those of M. litteratus.     

S-Gene Sequences and Genotype-Related Restriction Sites in Hepatitis B Virus Carriers in Turkey

Abstract Background: Although hepatitis B virus (HBV) infections are a major health problem in Turkey, there is little information on the genotype distribution of the virus. In this study, HBV genotypes were determined by DNA sequencing and restriction enzyme analysis of the S gene. Materials and Methods: The S genes of hepatitis B virus isolated from 23 chronically infected HBV-DNA-positive Turkish patients were amplified and directly sequenced. Nucleotide sequences were then aligned with reference isolates from the GenBank database and subjected to phylogenetic analysis. HBsAg subtype-specific aminoacid substitutions at codons 122, 127, 134, 159, and 160 were analyzed using translated sequences. The amplified products were also subjected to restriction enzyme analysis using endonucleases Mva I, Rsa I, and Hinf I. Results: Phylogenetic analysis showed clustering of all samples with genotype D sequences. The mean intragroup divergence was 0.95% (range 0.00%–4.00%). All isolates were ayw2, as predicted from translated sequences. The results of the restriction enzyme analysis of the samples were consistent with the phylogenetic analysis. Conclusion: Our data complement the findings that genotype D viruses are prevalent in the Turkish population. Being rapid and inexpensive, restriction enzyme analysis described in this study should be useful for large-scale epidemiological analysis of HBV infections.     

Molecular survey of rodent-borne Trypanosoma in Niger with special emphasis on T. lewisi imported by invasive black rats

Invading rodent species can harbor parasites with potential transmission to native rodents and/or humans. To investigate trypanosomes prevalence in rodents, the spleen of 76 rodents from Niger identified by their karyotype was used as a DNA source for Trypanosoma detection using a newly developed qPCR assay. Of the invasive black rat, Rattus rattus, 71% (10/14) were PCR positive as well as 6% (4/62) of native African rodents. Sequences of ∼400 bp of the SSU rDNA gene identified phylogenetically close Trypanosoma lineages. Trypanosoma lewisi was present in all positive black rats and the sequences displayed 100% similarity with T. lewisi-infected humans in Senegal. T. lewisi was also detected in one Acomys johannis, suggesting a possible transmission to native species. In addition to improved knowledge of Trypanosoma diversity in rodents, our data underscore the introduction of the potentially pathogenic T. lewisi kinetoplastid through the human-mediated invasion of black rats all over West Africa.     

Clinical Relevance of cagE Gene from Helicobacter pylori Strains in Japan

It has been reported that H. pylori-containing cagE was associated with duodenal ulcer. The aims of the present study were to clarify the association between the cagE gene and clinical outcome and to analyze the relationship between the cagE gene and two other virulence factors—cagA and vacA—in two areas in Japan (Fukui and Okinawa) where the prevalence of duodenal ulcer and gastric cancer risk are quite different. Eighty of 81 isolates possessed the cagE gene, and all isolates possessed the cagA gene. The vacA genotype s1c/m1 was a major genotype in both areas in Japan. There was no significant association between cagE, cagA status, or vacA genotype and clinical outcome. Phylogenetic analysis of the cagE gene indicated that most Japanese isolates formed a different cluster from strains isolated in the West with an association with the vacA genotype. In conclusion, the strains with cagE, cagA, and the s1c/m1 genotype of vacA are predominant in Japan regardless of clinical outcome and construct a different phylogenetic cluster from those in the West.     

Trans-spliced leader addition to mRNAs in a cnidarian

A search of databases with the sequence from the 5' untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5' ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra. The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.