extfieldsofstudy: Medicine Biology

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Trypanosoma cruzi transmission in the wild and its most important reservoir hosts in Brazil

Trypanosoma cruzi (Kinetoplastea: Trypanosomatidae) infects all tissues of its hosts, which along with humans, include hundreds of mammalian species in the Americas. The epidemiology of T. cruzi has been changing in that currently the majority of the cases and/or outbreaks of Chagas disease occur by the ingestion of comestibles contaminated by T. cruzi metacyclic forms. These cases/outbreaks occur in distinct regional scenarios, mainly in the Amazon biome and are related to the local interaction mode of humans with their surroundings, as well as with the overall local ecological peculiarities. As trypanosomiasis caused by T. cruzi is primarily a zoonosis, understanding the variables that influences its transmission in the wild as well as the role played by the extant fauna in the maintenance of the parasite, is critical in establishing control measures. Here, we present the results of our studies of T. cruzi infection of free ranging wild mammalian fauna in the five biomes of Brazil, a country of continental dimensions. From 1992 up to 2017, we examined a total of 6587 free-ranging non-volant wild mammal specimens. Our studies found that 17% of mammals were seropositive and 8% of all animals displayed positive hemocultures indicative of high parasitemia and, consequently, of infectivity potential. We observed that opossums, mainly Philander spp. and Didelphis spp., the coati Nasua nasua, the capuchin monkey Sapajus libidinosus and the golden lion tamarin Leontopithecus rosalia, were mammal taxa that demonstrated higher rates of positive hemocultures. Additionally, Didelphis spp. demonstrated to be a competent bioaccumulator of TcI diversity. Chiroptera were distinguished for hosting the greatest diversity of species and genotypes of Trypanosoma spp. Additionally the observation of the higher host range of some Trypanosoma spp., shows the need to reassess the ecology of representatives of the taxon. Altogether, our results showed that each locality, may display distinct enzootiological and epidemiological scenarios that must be taken into account when it comes to establishing control and/or clarification campaigns of the local population.

Background
The current rapid anthropogenic environmental changes, in addition to the uncontrolled human and animal migrations within an increasingly globalized world, act powerfully as parasite dispersers. This is the case of trypanosomiasis caused by T. cruzi that in humans can result in Chagas disease, currently considered a global health challenge [1].
Trypanosoma cruzi is a true generalist and highly successful parasite in that it is able to infect almost all cell types of hundreds of mammal species distributed from southern USA to the south of Argentina. This parasite species is transmitted by dozens of exclusively hematophagous triatomine bug species (insects of the order Hemiptera), in which the parasite establishes permanent infections. In its mammalian hosts, T. cruzi also establishes stable and long-lasting infections, that display peculiarities inherent to the different host species and even individuals and other multiple factors, such as the parasite subpopulations, infection route, host nutritional status and concomitant infection with distinct T. cruzi DTUs (discrete typing units) or other parasite taxa [2].
Trypanosomiasis caused by T. cruzi is primarily an ancient enzootic parasitosis of wild mammals and its most probable main dispersion strategy in nature is the oral route, by predation on both infected vectors and mammals. Consequently, T. cruzi transmission in the wild is deeply immersed in trophic nets [2,3]. Additionally, the classical contaminative route by metacyclic forms eliminated in infected triatomine feces may frequently occur in animals in their shelters and nests. It is also probable that the contaminative transmission by metacyclic forms of the scent gland of didelphid marsupials [4] represents an efficient transmission mechanism in the wild, but this has not been evaluated so far. This transmission route could have been important in the wild before hemipterans developed hematophagic habits [5]. The competence to infect all mammalian nucleated cells in any tissue as well as the diverse infectious forms, also represent a powerful mechanism of the maintenance of T. cruzi in nature. In addition to these evolutionary advantages, T. cruzi also displays a huge biological plasticity, probably as the result of its impressive biological, biochemical and molecular heterogeneity. Understanding the origin of T. cruzi heterogeneity still represents a challenge: although the population structure of T. cruzi is mainly clonal, mitochondrial introgression and hybridization events occur intra and inter-T. cruzi DTUs [6]. The frequency with which these phenomena occur in nature and their importance as promoters of T. cruzi diversity has been a highly discussed issue for decades. Currently, the set of these T. cruzi subpopulations were subdivided into seven taxonomic units or DTUs, namely TcI-TcVI and Tcbat. The latter, formerly assumed as restricted to bats, has been described infecting pre-Columbian human mummies [7,8]. The attempts that have been made to associate a given subpopulation of the parasite with the distinct forms of human disease, the ecology of its wildlife host species, the biome of its occurrence and vector species have not yet resulted in conclusive associations. If any association exists between a given parasite subpopulation and specific symptoms of the disease or reservoir, only long-term studies of a representative number of T. cruzi isolates of representative mammalian species of the different biomes and habitats will establish this. Currently, however, studies have been conducted with samples that are obtained usually on only a single occasion from a single infected individual. These samples are then subjected to selective pressures inherent to isolation and maintenance methods. In addition, representative T. cruzi isolates of representative mammalian species present in all habitats where the parasite occurs are lacking. Altogether, we are sure that these gaps result in huge biases.

After all, what constitutes a reservoir?
Defining what constitutes a reservoir is a practical and theoretical challenge, and there are currently many definitions [9,10]. Our concept of a reservoir is based on that proposed by Ashford [11], according to which a reservoir, or rather a reservoir system, is the species or the assemblage of species that support the maintenance of a parasite in nature in a sustainable way in a given space scale. Actually, among the several definitions, this seems the best-fit for a complex taxon as in the case of T. cruzi. Indeed, this multi-host parasite establishes peculiar and dynamic interaction patterns with each of its hundreds of host species, which therefore play different roles in the parasite transmission network of the different habitats and biomes of its occurrence.
The host's capacity of being infective to its vectors, under natural conditions, depends on several factors, the most important being the concentration of parasites in the mammalian host's blood (i.e. the parasitemia). That is, a mammalian host displays its maximum infectivity potential during the period in which it maintains a sufficient amount of circulating parasites to be ingested during the blood meal of the vector, ensuring its dispersion and maintenance in nature. The periods of high parasitemia vary among host species and individuals. The currently known variables that determine the duration of parasitemia in a given animal species (or even individual) include at least parasite genotype, nutritional status, infection route and concomitant infection. A combinatorial analysis of all the variables might result in an infinite number of possibilities. The competence of a mammalian host in infecting vectors was elegantly termed by Brunner et al. [12] as "realized reservoir competence".
Among the points that should be taken into account when evaluating a given mammalian species as a reservoir, is its precise taxonomic classification. In fact, even cryptic species may present significant differences of the course of the infection with the same species of parasite as is the case of Thrichomys laurentius, a caviomorph rodent species of the semi-arid caatinga in northeast Brazil, and T. fosteri, a species from the Pantanal, a flood plain region in west-central Brazil. Experimental infections with T. cruzi in these two rodent species showed that T. fosteri demonstrated a significantly higher and long-lasting parasitemia and much more severe histopathological lesions [13]. These rodent species, which are morphologically absolutely indistinguishable, demonstrated to manage T. cruzi infection in totally different ways and consequently play a very different role in the maintenance of T. cruzi in the natural environment, as was later demonstrated [14,15].
Evaluating the infectivity potential of a mammal species is a challenging task since it depends on a long-term follow-up of naturally or experimentally infected animals. In the latter case, the great challenge is the difficulty in rearing and maintaining wild mammal species in animal facilities. Experimental infections in conventional laboratory animal models are not representative and do not mimic infection conditions in the wild. In order to fully understand a reservoir system, it is important within the area of study to take an inventory of the local species, to analyse their population structure and to assess the taxonomic and beta diversity of an area.
One of the major obstacles to the study of wild reservoirs is the lack of species-specific reagents for serological tests. These tests are indispensable because they detect infected animals which have low, undetectable parasitemia, as shown by negative parasitological tests, i.e. low infectivity potential. Ideally two serological tests and two parasitological tests with distinct sensitivity should be employed.
In this review article, we summarize the data concerning wild reservoirs of T. cruzi collected during the last 20 years by our research group. We used data exclusively collected by our group as a way of making these data comparable. Moreover, we used the same techniques that we employed after establishing standard operating procedures (SOP) in our laboratory (Labtrip). The technical team, if renewed, received proper training by our staff before assuming any procedure as a way to maintain quality and repeatability. Thus, the methods employed in all steps were the same and are, therefore, comparable. In short: (i) blood cultures were performed in two tubes with 300 μl of blood in each that were examined fortnightly for up to five months; (ii) serology was performed through indirect immunofluorescent assay tests (IFAT), according to Camargo [16] Epimastigotes of T. cruzi, strains F (DTU TcI) and Y (DTU TcII) were grown in NNN/LIT medium to exponential phase and subsequently washed. Whole epimastigotes of each strain were mixed in equal proportions and employed as antigen in the IFAT assays. The reaction was revealed by species or specific family conjugate]; and (iii) molecular characterization at the DTU-level by means of two algorithms, mini-exon with RFLP and, more recently, nested-PCR for the 18S gene followed by sequence analysis. In this sense, all bat samples, as well as original isolates from other mammals not previously characterized in DTUs level, were recharacterized by the nested PCR protocol [17]. The complete methodology, as well as some of the results that were previously published, are described elsewhere [14,15,[18][19][20][21][22][23][24][25][26][27][28][29][30].
The infectivity potential, however, is not taxon-inherent and a fixed trait, as already mentioned above. A four-year study of the sylvatic transmission cycles of T. cruzi, performed in Argentina, concluded that cingulate and marsupials other than the genus Didelphis spp. displayed higher infectivity potential in comparison to Didelphis albiventris, classically described as the main T. cruzi reservoir [31]. Concerning domestic animals, dogs in Santiago del Estero (Argentina) demonstrated to be highly infective [32] in contrast to dogs in Brazil that rarely display high parasitemia [33,34].
The T. cruzi enzootic transmission cycle and its idiosyncrasies Trypanosoma cruzi enzootic transmission has been demonstrated to occur all over Brazil. Table 1 shows that 17% of the 6431 examined wild mammals had been exposed to T. cruzi infection as revealed by serological methods. Concerning Chiroptera, given their particularities, besides being the ancestral hosts and harboring numerous species of the T. cruzi clade, data referring this taxon are being presented in a separate table.
Positive hemocultures, i.e. infectivity potential, was observed in 8% of all 6587 examined animals distributed in all biomes. These data suggest that only 8% of individuals with infective competence are sufficient to maintain the widely dispersed and complex cycle of T. cruzi transmission. The lack of species-specific reagents impaired the performance of serological tests of all mammalian taxa. Even so, the sample of individuals that we were able to test was expressive and showed that 17% of them were seroreagent for T. cruzi showing that the infection of the majority of the infected mammals is expressed by positive serology (χ 2 = 197.36, df = 1, P < 0.0001). It is worth noting that infected animals were distributed in all biomes. The rate (percentage) of mammals displaying positive blood cultures was considerably higher in the Amazon in comparison to the other biomes (Table 1).
All mammalian orders (Artiodactyla, Chiroptera, Primates, Carnivora, Rodentia, Cingulata, Pilosa and Didelphimorphia) demonstrated to be involved in the transmission cycle of T. cruzi to a greater or lesser degree and in distinct time-and space scales (Tables 2, 3, 4, 5, 6 and 7). Thus, the lowest number of infected mammals has been observed in the Cerrado, contrasting with the Amazon biome, where T. cruzi infection rates were the highest. The following taxa may be considered as key reservoir taxa due to the higher rates of parasitemia (as expressed by positive hemocultures) and competence in maintaining a higher diversity of T. cruzi DTUs, as well as other Trypanosoma species: Primates, Didelphimorphia, Chiroptera, and the Carnivora species Nasua nasua. Mixed T. cruzi DTU infections are frequent, notably in generalist (concerning habitats and feeding habits) mammal species [2]. That means that, besides environmental conditions (habitats, climate, anthropogenic influence, etc.) both diversity and faunal composition are indicators of a more or less robust T. cruzi transmission cycle in a given area.
The T. cruzi infection rate of free-living mammals in the Amazon biome was significantly higher than observed in other biomes, both in relation to blood cultures and serology (IFAT). Free living mammals of Caatinga, Pantanal and Atlantic forest displayed comparable rates of positive hemocultures (Table 1; P < 0.0001). Moreover, the distribution of T. cruzi DTUs in Brazilian biomes is apparently non-homogeneous. As an example, the DTU TcII that was rarely found in the Amazon and even then, in localities that are distant from each other ( Table 2). This observation raises the question of how is a T. cruzi DTU that is only sporadically isolated, maintained and dispersed in the natural environment? Actually, there is still much to learn about the ecology of T. cruzi and its DTUs.
In the Cerrado, most trapped and examined mammals were rodents (54.6%) ( Table 5). Primates accounted for only 2.3% of the examined mammals, while marsupials and carnivores constituted 27.4 and 15.6% of sampled animals, respectively (Table 5). Only 2% of all examined mammals displayed positive hemocultures in this biome, which was statiscally different from other biomes (Table 1; It is worth noting that although the number of examined mammals and study areas is large, these are still not representative considering the huge diversity and number of the extant mammalian fauna and the huge ecological diversity and dimensions of the country. It was not possible for our group to test all of the examined wild mammal species by serological methods due to the lack of species-specific conjugates and/or insufficient amount of serum. Actually, blood cannot be collected from very small-sized animals in sufficient quantities for blood cultures (one pair of tubes with 300 μl each) and serology (50 μl sera).

Trypanosoma cruzi transmission among mammals with low parasitemia
In the Pampa biome, 14% of the animals demonstrated positive serology but none displayed positive blood culture ( Table 6). The questions that immediately arise are: how is transmission possible as it depends on high parasitaemia, and what is the strategy that ensures the permanence of the parasite in this scenario? In contrast, in the Amazon, 22% of mammals including those using all forest strata presented high parasitaemia expressed by positive blood cultures in a classical epizootic scenario ( Table 2). A hypothesis we favor is that the mammal fauna in the Pampa display only short periods of high parasitaemia, i.e. the transmission window of these individuals is narrow, and it is the assemblage of mammals at different stages of infection that ensures the persistence of T. cruzi in nature [2]. In areas in which mammalian fauna display low parasitemia, the transmission of T. cruzi may also rely on ecological features of hosts and triatomine, e.g. in higher host-vector contact as is the case of nesting animal species such as coatis, hoary foxes and golden lion tamarins.
The overall low T. cruzi infection rates observed in the order Rodentia that are highly susceptible to experimental infection suggest that these mammals play a secondary role as reservoirs in the wild (Tables 2, 3, 4, 5, 6, 7 and 8). This was especially striking in the Cerrado   biome, as expressed by their low infection rates (3.7% of positive serological tests and 0.7% of positive hemocultures) ( Table 5). Two hypotheses may explain these findings: (i) the infected animals do not resist the infection and die; or (ii) rodents are not exposed to infection in the studied Cerrado regions. Finally, these findings are similar to what was observed in the Pantanal biome [15] and support the hypothesis that, in fact, wild rodents only exceptionally play an important role as T. cruzi reservoirs. The miscellaneous, focal and aggregated character of the T. cruzi transmission cycle in the wild environment can be observed in two biomes. In the Atlantic Forest, of the 8.5% of the mammals showing positive blood cultures, 44.4% were constituted by two species of tamarins: 37.6% by Didelphis spp. and 10.7% by Philander spp. Overall, in the Atlantic Forest, only 0.35% of the rodents displayed positive blood cultures (Table 7). In the Caatinga, we observed one rodent species (Rattus rattus) with high rates of positive blood cultures (21%, Table 3), showing how important it is to look at every forest fragment as a singularity. Transmission patterns may change in time as revealed by studies of Trypanosoma spp. infection in coatis of a Pantanal region termed Nhecolandia. Initially, high rates of infection with T. evansi were observed (Table 4) [35]. The continued monitoring of this same coati population resulted in successful isolations of T. cruzi and also T. rangeli [30]. More recently,  coatis demonstrated to be again infected with T. evansi and to a lesser extent with T. cruzi or T. rangeli [2]. It is tempting to hypotethize that climatic alterations resulted in this process, but which ones? Trypanosoma evansi and T. cruzi display distinct transmission strategies T. evansi being mechanically transmitted by hematophagous dipterans and T. cruzi and T. rangeli, by triatomine vectors. All these data together show that host-parasite systems are complex and unpredictable. An example of independent transmission cycles occurring in the same forest fragment was observed in a rainforest fragment in the Atlantic Forest biome where a conservation program of the endangered golden lion tamarin (GLT) species, Leontophithecus rosalia, is maintained [25]. The study of T. cruzi infection of GLTs and sympatric mammals revealed that only GLTs were infected with T. cruzi DTU TcII and all other species of local mammals showed infections with TcI (Table 7) [25].
Of the examined infected mammals, those species that demonstrated the higher infectivity potential, as expressed by high rates of positive hemocultures throughout Brazilian biomes will be highlighted here. These mammal taxa are Didelphimorphia (Didelphis spp. and Philander spp.), procyonid carnivores (N. nasua) and primates (Table 8). It is worth mentioning that representatives of the Didelphimorphia displayed the highest infective competence in the Amazon, Atlantic Forest and Caatinga (along with primates in the first two biomes). In the Pantanal, N. nasua plays this role. Bats were distinguished for presenting the highest diversity of Trypanosoma spp. infection in simple and mixed infections and are discussed separately (Table 9).
Didelphimorphia (Didelphis spp. and Philander spp.) Marsupials comprise a mammal group which presents a peculiar reproduction strategy. These ancient mammals are an evolutionary success since they are extant, with little anatomical change since the Cretaceous [36]. The family Didelphidae includes all representatives of metatherian mammals or marsupials extant in the Americas. Didelphidae includes four subfamilies (Glironiinae, Caluromyniae, Hyladelphinae and Didelphinae), 19 genera and 95 species of which only four do not occur in South America [37].
The species within the genus Didelphis include the largest American marsupials. Didelphis spp. have a pair of scent glands adjacent to the cloaca that are a suitable niche for the multiplication of T. cruzi as epimastigote forms and for its differentiation to infective metacyclic trypomastigotes [4]. Didelphid marsupials have a very short gestation time (in Didelphidae, delivery occurs on average after 13 days of gestation). Individuals are born in an almost embryonic stage; the newborns do not have obvious eyes, ears or hind legs. They are absolutely hairless, and their mouth is a small hole through which the newborn will adhere to a nipple, and to which it will remain attached and totally dependent for six to eight weeks. Nevertheless, the newborn marsupial must climb from the womb without maternal aid to reach the pouch and adhere to a nipple. At this stage, and for the next several weeks, the newborn is totally dependent on the marsupium, and if removed will not survive. Despite the close and long contact between the newborn with the mother, in experimental conditions, we never observed neonatal transmission of T. cruzi in D. aurita [38].
Often described as essentially arboreal, Didelphis spp. use with reasonable frequency the soil, the understory, and the sub-canopy/canopy strata. However, the use of vertical space is not a fixed trait; it depends on the population structure, in that competition between young and adult animals may determine the level of occupation of the vertical space. Interspecific competition may also determine the use of the vertical space by this taxon [39]. In the wild, Didelphis spp. also use tree hollows, wood piles, palm crown and other locations for shelter, all of which are excellent habitats for triatomine species. Didelphidae are classically described as solitary animals with social interactions restricted to the mating season and to young pouch animals. However, this paradigm is being challenged by recent observations suggesting that these animals may display a social behavior pattern [40].  Didelphid marsupials are nocturnal, nomadic (mainly the males) and are agile climbers but quite clumsy when on the ground. Didelphis spp. can become infected orally (preying on small mammals or triatomines); they display high infectivity potential mainly for TcI (with high levels of parasitaemia almost indefinitely) and are able to infect other mammals directly in antagonistic situations as T. cruzi could be present in the secretions from the scent glands that are released by stress. Trypanosoma cruzi is not homogeneously distributed in the lumen of the scent glands. The epimastigotes are found along the glandular epithelium that is coated by hyaluronic acid. The metacyclic forms are found mainly in the middle of the lumen of the glands and metacyclogenesis occurs regardless of adhesion [41]. This ensures the preferential elimination of infectious trypomastigotes when scent glands are used. All these characteristics have, as a consequence, that didelphids are exposed to all of the T. cruzi transmission cycles that occur in these habitats.
Didelphis spp. are extremely habitat and food resilient and easily adapt to peri-domestic areas. There, they feed on human food remains and also serve as food for humans. This is especially the case among the riverine settlements in the Amazon rainforest. Besides being an important source of protein, Didelphis spp. are also used for medicinal purposes by these populations; the meat is described as tasty and of good quality [42]. The preparation of opossums without the proper care of hygiene also represents a T. cruzi infection risk to humans.
Marsupials of the genus Philander are smaller than Didelphis spp. Philander spp. are extremely agile on the ground and also display remarkable climbing ability. They are also described as the most aggressive didelphids. These opossums are exclusively nocturnal and, although often caught on the soil, they are also arboreal. They live in dense forests or underbrush where they build their nests of leaves but may also eventually use branches or fallen trunks as shelter. Their preferred environments appear to be gallery forests or other more humid environmetns, where they are often found. Philander spp. display quite eclectic feeding habits and predate both on insects and small vertebrates, including mammals. They also feed on fruits and eggs.
Due to their nomadic character, generalist feeding and housing habits, Philander spp. and Didelphis spp. are exposed to T. cruzi transmission cycles occurring in all forest strata in the wild. Interestingly, species of these two genera of marsupials handle experimental infections with T. cruzi differently. Thus, Didelphis spp. control to subpatent levels infections with the Y strain of T. cruzi (TcII) but maintain indefinitely high parasitaemias (expressed by positive blood cultures) in infections with TcI strains. In contrast, Philander spp. maintain high parasitaemia when inoculated with strain Y (TcII) and control the parasitemia with TcI strains [43]. TcI and TcII are, however, found in mixed infections, and this is the most common and most widely dispersed combination that we found infecting both mammals and vectors [2].
As shown in Table 8, Didelphis spp. and Philander spp. demonstrated high rates of positive blood cultures in the Atlantic Forest and the Amazon. In the semi-arid region of the north-eastern Brazil. Didelphis spp. act as an important reservoir. In the Pantanal, in addition to being uninfected, the relative abundance of these two species was very low. In this immense flood plain region, the primary reservoir of T. cruzi is the procyonid N. nasua.
A very interesting and puzzling finding was reported by Dario et al. [44] who observed one Monodelphis americana (Didelphimorphia) specimen infected simultaneously with T. cascavelli, T. dionisii and Trypanosoma sp. Trypanosoma cascavelli was originally described infecting a Crotalus durissus terrificus [45]. There are three interesting aspects here: (i) the infection of a marsupial with a Trypanosoma species associated exclusively to bats (T. dionisii); (ii) a Trypanosoma species that is able to infect both a mammal and a reptile in a mixed infection with T. dionisii; and (iii) the presence of a still unknown taxonomic unit of Trypanosoma sp. Concerning the second   The infection of marsupials with T. dionisii reinforces this species as a generalist species, since this parasite was already found infecting humans [18]. Actually, humans, bats and marsupials are phylogenetically very distant taxa.

The coati, N. nasua, and other carnivores
Working with carnivores is not easy and requires a complex infrastructure and well-trained experienced professionals. This might explain why most studies of reservoirs of T. cruzi do not include representatives of this group. Most carnivores are adapted to terrestrial life, but they may also be excellent swimmers and climbers. Carnivores use large living areas, are at the top of the food chain and are talented hunters. Consequently, it is very probable that these animals are more prone to infection via the oral route. Not all carnivores are flesh eaters sensu stricto. There are opportunistic carnivore species that also feed on insects and fruits. Only the  All carnivores, including domestic dogs and cats, may be involved in the transmission cycle of T. cruzi [32,33,46]. Concerning wild carnivores, infection with T. cruzi was demonstrated in 15 Brazilian Neotropical wild carnivore species [3]. A link between diet peculiarities and T. cruzi infection rates in these taxa could also be observed. Thus, insectivore canids and hypercarnivores (those that include flesh as more than 70% of their diet) displayed higher T. cruzi infection rates than other carnivores with other types of diet. These findings confirmed that T. cruzi transmission cycles are deeply immersed in the trophic nets. Additionally, differences in infectivity potential across the carnivore species have also been described the Procyonids, mainly N. nasua, N. narica and Procyon lotor, being the carnivore species that display the more expressive infectiveness potential as expressed by high and long-lasting parasitemias [3,27,[47][48][49]. Note that Procyonids are omnivores that also feed on both insects and small mammals [50], a trait that enhances their chances of infection with the diverse T. cruzi DTUs.
Coatis are scansorial, diurnal and generalist procyonid carnivores that use both the arboreal and terrestrial strata [50,51]. They are peculiar mammals in that they construct and use arboreal nests for resting and birthing [52]. One individual may use several nests during its lifespan, or more than one individual or even a whole social group may share the same resting nest. This behavior increases the probability of encounter between coatis and triatomines, since bugs also use these nests as shelter, consequently enhancing T. cruzi transmission chances. Nasua nasua are organized in social groups that include mainly females and young, still immature individuals, and only a few adult males [53]. The birthing nests are constructed by the females that give birth to up to seven pups. In these nests, which are larger than the resting nests, the female remains caring for her cubs for about two months [51,52].
Encounter rates between free-ranging mammals and vectors are one of the most important and challenging variables to be determined. Actually, animal shelters and nests may be used successively, by several species of living organisms, including triatomine vectors of T. cruzi. Coatis arboreal nests offer shelter for several other animal taxa, forming genuine and complex biocenoses. On dissection, these nests can be found to contain ants, lizards, beetles and triatomines. Arboreal nests of coatis have also been demonstrated to be suitable habitats for Rhodnius stalii and Triatoma sordida, which were described living in sympatry, in a same arboreal coati's nest. Even a terrestrial rodent has been described visiting a coati's nest [54]. Probably, this rodent used fallen trunks or vines to reach the nest that was over ten meters above the ground. This report shows that the detection of a particular T cruzi DTU in an individual mammalian host species does not necessarily mean that there is an association between the DTU and that species or forest stratum.
During a study of coati ecology, Santos et al. [55] collected Triatoma sordida (n = 13) and Rhodnius stali (n = 2) from seven coati resting nests. Additionally, 21 nymphs of all stages were collected from these nests, confirming these as suitable habitats for triatomines. Seventeen isolates of T. cruzi derived from insects of four coati nests were obtained, mostly TcI. One single insect was found infected with TcIII, and four individuals displayed mixed infections with TcI and TcIII. The use of these nests by coatis and other animals provides a suitable habitat for the maintenance of the triatome colonies since sufficient food source and shelter is offered.   As avid insect predators [50], coatis may acquire T. cruzi infections via the oral route, which is recognized as highly efficient [56]. In fact, the dense fur of the majority of wild mammal species most probably acts as a barrier against the infection via the contaminative route. Nasua nasua has been described as a competent T. cruzi reservoir due to their long-lasting parasitemia demonstrated by positive hemocultures. Furthermore, N. nasua has been shown to be able to maintain the main genotypes of T. cruzi (TcI and TcII) in mixed or single infections (Table 4). This ability to maintain T. cruzi populations, together with their generalist character, suggests that the coatis may be exposed to many or all of the various transmission cycles of the parasite. Taken all together, this implies that this species may act as a hub of the T. cruzi transmission network and a bio-accumulator of T. cruzi subpopulations [3,27,30,47]. Also of interest is that Rocha et al. [3] observed T. cruzi isolates that did not fit into any of the six recognized DTUs, suggesting that there is still much to learn about the diversity of this parasite. Table 4 shows infection rates and T. cruzi genotypes observed in N. nasua from Pantanal region.
Primates: golden lion tamarins in the Atlantic Forest and Cebidae in the Amazon region New World primates are essentially arboreal, and some use their prehensile tail as a fifth limb. It is currently accepted that this taxon (as well as caviomorph rodents) Table 7 Trypanosoma cruzi natural infection in free-living wild mammals from the Atlantic Forest biome: taxonomic identification of the examined mammals, results of hemocultures, indirect immunofluorescence tests (IFAT), and characterization of parasites. In the case of insufficient blood for hemocultures and serological tests, we prioritized the blood cultures (Continued)      (Tables 2 and 7). Tamarins (Leontopithecus rosalia and L. chrysomelas) were found infected with T. cruzi in the Atlantic Costal Forest in both the southeast and the northeast regions, mainly with TcII, but also with TcI (Table 7).
Tamarins are diurnal primates that live in social groups of up to ten individuals. Leontopithecus rosalia, the golden lion tamarin (GLT), is an elegant and delicate primate; the adult weighs between 360 and 710 g, and 60 g is considered normal for a GLT puppy. They are endemic to the Atlantic Forest and are in serious danger of extinction. Tamarins can live up to 15 years and are essentially arboreal.
A ten-year follow-up of T. cruzi infection revealed that L. rosalia is able to maintain a stable and long-lasting T. cruzi TcII transmission focus. It is interesting to note that in the studied area, only L. rosalia showed infection with this DTU: the other mammals were infected only with TcI. Leontopithecus rosalia infection with TcI was also observed in the area that is made up of a complex of forest fragments designed for tamarin conservation. Still in the Atlantic Forest, but in the northeast of the country, another species of tamarin (L. chrysomelas) also showed high rates of infection with T. cruzi TcII [25]. Such a stable transmission cycle of T. cruzi DTU TcII is not frequently found in the wild environment. What is seen in nature is that TcII infects numerous species of wild mammals distributed in all biomes but at low rates of infection. Trypanosoma cruzi DTU TcI is strongly predominant among free-living wild mammals [2]. An interesting aspect shown by the study of T. cruzi infection in tamarins is that even in a small area (5500 ha), such as the forest fragment where these primates were studied, the T. cruzi infection distribution was aggregated [25].
The congenital infection of L. rosalia is probably not very frequent because T. cruzi infection was not observed in juveniles [25]. Possible sites of infection are the hollows of trees that serve as a night shelter for these animals. Each hollow is shared by a social group in a rotation system of the hollows used. Inside a hollow, animals sleep tightly curled and the younger animals are at the centre. A hollow tree might be a suitable habitat for triatomine bugs in that there is an abundant food supply offered by the tamarins. These hollows are high, deep in the trunks and feature a very narrow entrance. The oral route by ingestion of infected triatomine must also be an important route of infection in tamarins, even if triatomine bugs are not very attractive insects because of their scent glands. The simple act of chewing the insect may result in infection, even if the insect is later ejected. Studies on the health of GLTs infected with T. cruzi based on electrocardiograms, blood tests and electrophoretograms showed that GLTs appear to support the parasitization by T. cruzi without major damage [59].
High rates of T. cruzi infection with high parasitemia were also observed in Sapajus libidinosus (Cebidae) in the Amazon (Tables 2 and 8). This species is the only New World monkey that uses tools in the natural environment to break nuts. Sapajus spp. are strictly diurnal, display arboreal habits and spend most of their time foraging and moving through their life area (~300 ha) [60]. They are extremely social mammals that live in groups of up to 50 individuals, led by a larger, older male. They are also cooperative in raising young and finding food. Cebidas are long-living animals and sexual maturity occurs earlier in females (4-5 years) than in males (8-10 years). These  [61,62]. Cebids are difficult subjects for research and we did not have chance to conduct a long-term study. Concerning T. cruzi infection, S. libidinosus presented TcI in single and mixed infections with T. rangeli. Single infections with T. rangeli have been also observed ( Table 2).
All species of Sapajus are omnivorous, but their main diet consists of fruits and insects, in addition to small vertebrates including mammals, which means that the oral route must be important route of infection with T. cruzi in this primate group. Sapajus spp. are arboreal primates and trees are selected in part for food availability. They frequently use palm trees as places to sleep which also provide shelter to triatomine bugs; consequently, infection via the contaminative route during the night is also possible. Table 9 and Fig. 1 show the great diversity of Trypanosoma spp. infecting bats and that T. cruzi was the most abundant species of Trypanosoma (37%). Moreover, the richness of Trypanosoma spp. infecting this mammalian taxon is much higher than observed in any other mammalian host. The majority of positive hemocultures of bats revealed the infection by other Trypanosoma spp. and not T. cruzi. These findings show that bats' role as T. cruzi reservoirs was probably often overestimated at a time when there was still no easy access to sequencing tools. The use of even more sensitive and high-performance tools like NGS would certainly demonstrate even greater diversity of genotypes and species in single and mixed infections in bats. In addition to bats, marsupials have also been found infected with numerous species of Trypanosoma including T. dionisii, a species considered to be an exclusive parasite of bats until recently [44]. An important question concerns the result of such mixed infections in hosts: what are the consequences in the outcome in such mixed infections?

Chiroptera
Bats may be considered as Trypanosoma spp. bio-accumulator hosts. There are plenty of recent descriptions of new species and still undescribed taxonomic units of Trypanosoma spp. infecting chiropterans all over the world. Chiropterans' longevity and ability to fly, as well as their capacity in maintaining T. cruzi DTUs and other representatives of the T. cruzi clade, imply in that these mammals may act as important dispersers of these flagellates. Molyneux [63], and more recently Hamilton et al. [64] proposed bats as the ancestral hosts of the T. cruzi clade and that terrestrial mammals became infected with T. cruzi as a consequence of several spillover events of the bats trypanosomes. They termed this evolutive path as "the bat seeding hypothesis". This hypothesis is currently universally accepted based on the description of trypanosomes of the T. cruzi clade infecting African bat species in contrast to the low diversity of T. cruzi clade species other than T. cruzi infecting the American terrestrial mammals.
During the last two decades, we examined a total of 1219 bat specimens distributed in 76 genera and 94 species of five Brazilian biomes (Table 9). High infectivity potential for Trypanosoma spp., as demonstrated by positive hemocultures, was observed in 14% of these mammals. From these, only 5% were infected with T. cruzi, in single or mixed infections. The remaining 9% were infected with other Trypanosoma species such as T. c. marinkellei, T. dionisii and T. rangeli (Fig. 1).
The highest rates of Trypanosoma spp. infections in bats were observed in the Amazon region (22.6% of positive hemocultures), followed by the Cerrado biome ( Table 9). As expected, a higher diversity of bats and Trypanosoma spp. species were observed in pristine forest areas in comparison to already altered forest patches. In this sense, a higher variety of bat species was observed in the Amazon in comparison to the Atlantic Forest. This issue is especially striking considering that in the Atlantic Forest the number of examined individuals was almost double that of Amazon (Table 9). Among the infected bats, 62 specimens displayed single or mixed T. cruzi DTU infections and the majority of the chiropterans were infected with TcI, but other T. cruzi DTUs also may infect bats, demonstrating that these animals are rather resilient to T. cruzi genotypes. We did not find T. cruzi TcII infecting Chiroptera, and these data raise doubts on what was previously known about bat Trypanosoma spp. infections and forces us to rethink the subject.
Of the bat species, members of the genera Artibeus, Carollia and Phillostomus (Phyllostomidae) exhibited the highest rates of infection with the different Trypanosoma species. These three genera include generalist species that feed on fruits, but also pollen and insects. Bats are important seed dispersers and probably become infected with T. cruzi through the contaminative and oral routes, the latter by ingestion of infected triatomine bugs and mice, as already demonstrated experimentally [65].
Next generation sequencing (NGS) demonstrated to be a promising tool to answer questions concerning Trypanosoma specificity, ecology and phylogeny. The possibility of detecting trypanosomatids that are not cultivable in axenic media, as well as the possibility of bypassing the selective forces inherent to isolation methods, are highly informative. Dario et al. [66] conducted a study by metabarcoding DNA samples extracted from blood samples of 34 bats of 13 species captured in Atlantic Forest fragments of Espírito Santo state (southeast region of Brazil). The resulting amplicon sequences could be clustered in 14 operational taxonomic units (OTUs). Five OTUs were identified as Trypanosoma cruzi TcI, T. cruzi TcIII/V, T. cruzi marinkellei, T. rangeli and T. dionisii, very common trypanosomatid species in Neotropical bats. However, seven clusters demonstrated to be novel genotypes related to the Neotropical bat genotypes. One OTU was identified as a reptile trypanosome and, surprisingly, another OTU was identified as Bodo saltans, a free-living Kinetoplastea.
The majority of the bat blood samples demonstrated mixed Trypanosoma spp. infections and also mixed OTUs infections by up to eight OTUs. The massive rates of mixed infections are certainly not exceptional in nature and deserve further studies as to evaluate their influence on the course of infection. Actually, these mixed infections cannot be overlooked.

Conclusions
The set of observations presented here is the most complete and comprehensive study on reservoirs and wild hosts of Trypanosoma cruzi. The 20 years of data collection and analyses show that there is no association of Trypanosoma cruzi DTU with mammalian host species in their habitat or the biome of their occurrence. In addition, it was possible to conclude that T. cruzi is transmitted and remains in the wild even if most of the individuals of the wild mammalian species infected have low parasitemias. Mixed Trypanosoma cruzi DTUs and Trypanosoma spp. are very common in the wild, and chiropterans and marsupials are the mammals that present the highest diversity of genotypes and infection rates of T. cruzi and other species of the genus Trypanosoma spp.


extfieldsofstudy: Chemistry Medicine

text:
Enhancing atrial‐specific gene expression using a calsequestrin cis‐regulatory module 4 with a sarcolipin promoter

Abstract Background Cardiac gene therapy using the adeno‐associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off‐target localization. To overcome this, studies have applied cardiac‐specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial‐specific gene expression, an enhancer calsequestrin cis‐regulatory module 4 (CRM4) and the highly atrial‐specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. Methods To observe expression and bio‐distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. Results The CMV promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module SLN combination showed enhanced atrial expression and a minimized off‐target effect even when compared with the individual SLN promoter. Conclusions For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra‐atrial expression.

been managed previously through transcriptional targeting. 8 Because the virus regulates transgene production through its promoter, promoter alterations have been used to control gene expression in various organs to avoid off-target effects. To overcome this barrier, cardiac-specific promoters have been applied.
Current cardiac-specific promoters investigated include α-myosin heavy chain, myosin light chain, enhanced myosin light chain and cardiac troponin T promoters. [9][10][11][12][13][14] Among the promoters investigated, some have shown chamber specificity. Because the atrium and ventricle differ morphologically, functionally and are molecularly distinct, pathophysiology also varies in cardiac diseases. Thus, using a cardiac-specific promoter for universal transgene expression in the heart in certain disease states can also affect healthy tissues negatively. Accordingly, the use of a specific promoter is critical in chamber-specific diseases.
Currently, several promoters have been observed to regulate cardiac chamber-specific gene expression. Among these are the atrial myosin light chain-2a, slow myosin heavy chain-3, atrial natriuretic factor and sarcolipin (SLN) for the atrium and the myosin light chain-2V for the ventricle. 15 The uniqueness of these promoters has been exploited in other methods in addition to improving specificity, such as allowing for cardiomyocytes generated from human-induced pluripotent stem cells to be separated by subtype-specific promoter-driven action potentials. 16 However, the application of these promoters to drive chamber-specific transgene expression has been limited as a result of the compromised efficiency of these promoters. To apply these promoters for use in gene therapy, the efficient transduction of these promoters is essential.
To overcome this, two sequences established previously were used to make an atrial-specific promoter with enhanced expression. We approached this issue by taking advantage the atrial-specific promoter of SLN. The SLN promoter was chosen because the SLN protein is characteristically expressed in the atrial chamber of the heart. Furthermore, it has been observed to change dependent on disease states. Sarcolipin was found to be up-regulated in rodent models of congenital heart disease and in patients with preserved left ventricular ejection fraction and chronic isolated mitral regurgitation. 17,18 By contrast, SLN was found to be down-regulated in patients with chronic atrial fibrillation. 19 In Duchenne muscular dystrophy mice, a reduction in SLN expression was found to mitigate associated cardiomyopathy. 20 Alone, the SLN promoter activity is unsubstantial compared to that of ubiquitous promoters. Thus, the addition of calsequestrin 2 cis-regulatory module 4 (CRM4), a cardiomyocyte-specific enhancer, which showed superior activity in the heart, was used to heighten transgene expression of SLN within the heart. 21 In addition to cardiac tissue, high SLN levels are found in the skeletal muscle and diaphragm. 22

| pTR.SLN.Luc
The shortened SLN promoter used was 1029 bp. This was identified as the primary core promoter sequence by removal of polyadenylation sequence from a 1253-bp human SLN promoter pur-

| Generation of AAV
The following self-complementary AAV (serotype 9) constructs were

| Transverse aortic constriction (TAC)
Mice were anesthetized with a solution mixture of 95 mg kg -1 ketamine and 5 mg kg -1 xylazine administered via intraperitoneal injection.
Mice were ventilated with a tidal volume of 0.2 mL and a respiratory rate of 110 breaths per minute (Harvard Apparatus). A longitudinal incision of 2-3 mm was made in the proximal sternum to allow visualization of the aortic arch. The transverse aortic arch was ligated between the innominate and left common carotid arteries with an overlaid 27-gauge needle. The needle was then immediately removed, leaving a discrete region of constriction.

| Fluorescence imaging
Mouse heart tissues were cut in a sagittal plane and fixed in 4% paraformaldehyde for 24 hours and moved to 20% sucrose for cryoprotection for 24 hours before being cryopreserved to OCT compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) at -80°C. Tissues were sectioned into 6-μm thick slices (CM 3050S; Leica, Wetzlar, Germany). The slides were dehydrated in 100% ethanol for 10 minutes and dipped in two changes for xylene. Slides were sealed with glass slides using mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield, Burlingame, CA, USA) and analyzed using a

| Western blot analysis
Tissue homogenates were lysed using RIPA buffer (Thermo Scientific) with protease inhibitor cocktail (Sigma-Aldrich). Protein quantification was conducted using Pierce BCA Protein Assay Kit (Thermo Scientific).
Proteins were resolved on 10% SDS-PAGE gels followed by transfer to nitrocellulose membrane (Bio-Rad, Munchen, Germany). Antibodies

| Statistical analysis
Statistical analyses were performed using a two-tailed Student's t-test, with significant differences demonstrated as appropriate. Data are reported as the mean ± SD. sequence significantly improved SLN promoter activity (p < 0.005).  A luciferase assay was conducted to determine bio-distribution. Luciferase activity was normalized to background values. Statistical significance was measured with a two-tailed Student's t-test and significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.005. Bar graphs represent the mean ± SD (n = 4) (p < 0.005), skeletal muscle (p < 0.01), diaphragm (p < 0.005), lung (p < 0.05), liver (p < 0.005) and kidney (p < 0.005) compared to control.

| EGFP
The luciferase was exchanged with an EGFP reporter gene: AAV9-

Protein analysis
Bio-distribution was observed in the atrium, ventricle, skeletal muscle, diaphragm, brain, lung, liver and kidney with western blotting using
Quantified luciferase values are shown in the table in supporting information (Table S1).   25 However, the promoter used to drive expression of the gene of interest often causes undesired activity in off-target tissues, especially in hepatic tissue. 7 Thus, cardiac-specific promoters have been identified to improve cardiac specificity. [9][10][11][12][13][14][15][16] Even within these promoters, chamber specificity has been noted. 14,15,23 Because the atrium and ventricle have individual functions, mechanisms and molecular signatures, pathophysiology varies in cardiac diseases as well.

Fluorescence imaging
Thus, using a chamber-specific promoter may be a better option than a global cardiac transgene expressing promoter. However, the transduction efficiency of chamber-specific promoters is often compromised as a result of poor activity. 10 With the current research on transcriptional regulation, we designed a combined a novel combination with synergistic effects to obtain enhanced atrial specificity. 21 Various enhancer sequences have been reviewed, 26-30 although FIGURE 5 mRNA analysis validated EGFP gene expression profiles. In vivo gene expression was observed 3 weeks post tail vein delivery of control and EGFP containing CMV, SLN and CRM4.SLN vectors. qRT-PCR analysis was conducted to determine bio-distribution with EGFP and normalized with 18S. Statistical significance was measured with a two-tailed Student's t-test and significant differences are indicated: *p < 0.05, **p < 0.01, ***p < 0.005. Bar graphs represent the mean ± SD (n = 4)

FIGURE 6
Visualization of biodistribution. In vivo gene expression in the atrium and ventricle of control and EGFP containing CMV, SLN and CRM4.SLN vectors was observed by fluorescence imaging using a confocal microscope at 200× magnification. Bright field, DAPI, EGFP and merged images were taken under same exposure and normalization settings CRM4 was the most promising candidate as a result of its superior cardiac specificity. Thus, we have manipulated the promoter by adding CRM4, a cardiomyocyte-specific enhancer, to improve transcriptional regulation of SLN. 21 In the present study, we evaluated an atrial-specific promoter sequence enhanced by a cis-regulatory module in vitro and in vivo. Initially, to evaluate promoter efficiency, we observed expression in HL1 cells and confirmed that the CRM4 within the gene cassette significantly improved luciferase activity driven by the SLN promoter in atrial cells (p < 0.005) ( Figure 3A). Thus, we followed with in vivo bio-distribution studies in wild-type mice to quantify potential offtarget effects in six other tissues, including the skeletal muscle, diaphragm, brain, lung, liver and kidney. Skeletal muscle and diaphragm were investigated because high SLN expression was reported. 22,23 Other tissues were also measured to evaluate AAV9 off-target effects. [5][6][7] To compare gene expression levels under SLN and CRM4. SLN promoters, two reporter genes were used for assessment: luciferase and EGFP. The luciferase assay showed that SLN.Luc was not only expressed in the atrium, but also detected in the ventricle and liver.
On the other hand, the CRM4.SLN.Luc showed highly selective luciferase activity in the atrium compared to the ventricle and other tissues.
Strikingly, CRM4 significantly minimized off-target effects even compared to SLN promoter used alone.
For alternative verification for protein and mRNA analysis, EGFP was used as a supplementary reporter gene. Protein evaluation with western blotting supported the luciferase assay data, which showed that the CRM4.SLN.EGFP had exceptional atrial specificity and basal expression level in other tissues (Figure 4). The EGFP reporter gene offered additional insights by showing expression in skeletal muscle as well. Because SLN is also significantly expressed in skeletal muscle, expression was not unanticipated. However, in conjunction with the luciferase assay data, which showed expression in the atrium to be significantly stronger when all samples were compared in reference, skeletal expression was concluded to be lower than atrial expression. qRT-PCR data of the EGFP transferred mice also supported atrial-specific expression with CRM4.SLN.EGFP ( Figure 5). Inconsistencies between RNA and protein levels in certain tissues such as the lung, kidney and brain reflect that our acquired data for qRT-PCR showed relative values instead of actual mRNA. Furthermore, visualization of EGFP fluorescence was observed with sectioned tissue of the atrium and ventricle ( Figure 6). The strong correlation between the luciferase and EGFP reporter genes both supported that the CRM4.SLN combination enhanced atrial specificity. Furthermore, we tested whether specificity to the atrium was preserved at higher doses of CRM4.SLN.
Although, at lower dosages, EGFP was previously not observed in the liver, at higher dosages, expression in the liver occurred. Nevertheless, CRM4.SLN confers exclusive atrial specificity among these three promoters.
For future application of this construct, the CRM4.SLN can be used for atrial-specific gene therapy in disease models. Because sarcolipin expression is increased in heart failure states (Figure 1), the sarcolipin promoter in diseased states may be advantageous, similar to that of the ANF promoter. 11 Furthermore, current therapeutic gene transfer for atrial fibrillation is highly limited, such as direct injection to the right and left atrium or to the atrioventricular node for rate control. 31,32 However, these approaches can lead to tissue damage and inflammatory response. 33,34 The most effective reported method is the gene painting method, which uses a poloxamer gel, dilute trypsin and vector mixture to increase contact time and penetration. 35 However, this must be performed in open heart settings, which increases mortality risk. 36 The use of this construct can offer a non-invasive and technically simple approach for atrial-specific gene therapy. The CRM4 and SLN combination caused robust and highlyspecific atrial activity and is a promising method of targeting transcriptional mechanisms to improve atrial transgene transduction.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Cytochrome P450 Gene in Silkworm (Bombyx mori): A Review Article

To understand features of cytochrome P450 genes in silkworm, the advances in silkworm genome projects created a new genome wide analysis tool that enables us to seek cytochrome P450 genes constitutes and their diversities. This has provided not only an opportunity for divergence in their substrate specificity identification, but also for divergence in their regulatory attributes, such as differing patterns of induction, tissue specific expression, and developmental expression. However, different researchers so far made different efforts to identify number of cytochrome p450 genes in Bomboxy mori, they came up with slightly different numbers which ranges from 79 to 86 putative cytochrome p450 genes. Then, it urges us to undertake further investigate in order to arrive at certain consensuses putative cytochrome p450 genes number. This work is licensed under Creative Commons Attribution 4.0 License WJASS.MS.ID.000502. World Journal of Agriculture and Soil Science Volume 1-Issue 1 Citation: Endale H, Yuehua Z, Guodong Z. Cytochrome P450 Gene in Silkworm (Bombyx mori): A Review Article. World J Agri & Soil Sci. 1(1): 2018. WJASS.MS.ID.000502. DOI: 10.33552/WJASS.2018.01.000502. Page 2 of 2 genes can be assigned to one of four clades: CYP2, CYP3, CYP4 and the mitochondrial CYP clade. The CYP3 clande is subdivided again into the CYP6, CYP9, CYP28, CYP308–310, CYP317, CYP321, CYP324, CYP329, CYP332, CYP336–338 and CYP345–348 families. Indeed, Many CYP9 family members are known to participate in detoxification pathways associated with insecticide resistance. Hence, they are considered as key enzymes in order to visualize many metabolic pathways and based on these signals that enable to formulate desirable remedial actions[1, 10-11]. Having these in mind, this review aimed at P450s in silkworm species. Cytochrome P450 Genes in Bombyx Mori Based on the advances in silkworm genome projects, a new genome wide analysis of cytochrome P450s genes was performed by different scientists. Consequently, their finds revealed that Bombyx mori genome has not similar putative p450 gene numbers. That is Li and his colleagues were discovered that 86 putative P450s in silkworm genome, which are thought to belong to 32 subfamilies. Further, they mad a comparative genomic analysis with Drosophila melanogaster, then the result revealed that the two insects have some similar P450 distribution patterns but still have some obvious differences. Especially, the diverse distribution exists in 7 p450 subfamilies, which are CYP4A, CYP4C, CYP4D, CYP6A, CYP6AE, CYP6B and CYP9A [11]. But, Li and his colleagues in other study, they came up with different result of predication number of putative p450s which was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported [12]. On the contrary, Ai and his colleagues predicted that based on the same genome project and analysis of cytochrome P450 genes they obtained a total of 84 CYP-related sequences which could be classified into 26 families and 47 subfamilies according to standard nomenclature. According to their report out of total eighty four genes, seventy eight of them were appeared to be functional and six were probable pseudogenes. Furthermore, their result revealed that the distribution of Bombyx mori P450s in the genome depicted that most of them are tandem arranged on chromosomes, while merely 34 genes were present as singletons, with 8 clusters including 3 or more than 3 genes . On the other hand, they came up with B. mori and H. armigera CYP9A genes clustered in species-specific groups of three P450s, with the exception of the orthologous CYP9A22/CYP9A14 pair, which indicates that the gene expansions are independent species-specific events relative to the last common ancestor. As different phytophagous insects, B. mori and H. armigera encounter many predictably different chemistries and xenobiotics in their respective host plants and their distinct environments [3]. Hence, due to these dynamic and selective pressures on gene copies mechanisms exposed them to engage into a gradual modification which will create genes with specific functions than their precursors. In general, we infer that these gene evolution scenario will drive insect P450s to diversify rapidly.

Introduction
Cytochrome P450 monooxygenases (termed CYPs or P450s originated from spectrophotometric peak at wavelength of the absorption maximum of the enzyme at 450nm) are collective name for the heme-protein containing enzymes that constitute a ubiquitous of hydrophobic, cysteinato-heme enzymes. They are one of the oldest and largest super-family. For their enzymatic reaction a variety of small and large molecular substrates are utilized. In general, terminal oxidase enzymes in electron transfer chains, categorized as P450-containing systems [1][2][3][4].
As the means of oxidation, P450 uses molecular oxygen, inserts one of its oxygen atoms into a substrate, and reduces second oxygen to a water molecule, using two electrons that are provided by NAD(P)H via a reductase protein. Since only one of the two oxygen atoms, initially present in O 2 , remains in oxidized substrate which result in P450s as monooxygenases. Molecular oxygen, itself, is unreactive toward organic molecules at low temperatures either due to spin forbiddingness or to high barriers. Consequently, living systems mainly use enzymes that modify dioxygen to form capable of performing desired oxidation reaction. This modification can be achieved by metal-dependent oxygenases, like cytochromes P450 or non-heme metalloenzymes (e.g., methane monooxygenase), or by flavin-containing enzymes that do not possess a metal-based prosthetic group [4][5][6][7].
Indeed, P450s are important metabolic enzymes involved in metabolism not only of a wide range of endogenous compounds such as fatty acids, steroids, hormones or vitamins, but also exogenous substrates such as drugs, chemicals including environmental pollutants, such carcinogens as polycyclic aromatic hydrocarbons, and pesticides as well as plant toxics. Consequently, they are found virtually in all aerobic organisms, including organisms as diverse as in insects, plants, mammals, birds and bacteria [1,[5][6][7][8][9]. Recently, due to the advancement in genome sequencing technology a huge number of P450 enzymes (more than 21,000 sequences of P450 genes) are accessible in the P450 database from all kingdoms of life [1]. The diversify of sequences encoded by a multiplicity genes, which have been arisen by gene duplication and adaptive diversification results in functional versatility of the p450 superfamily i.e., gene duplication is basically a corner stone for evolution of gene family [3].
The crucial roles of P450s, in insects are synthesis and degradation of insect hormones including ecdysteroids and juvenoids and the detoxification or activation of such chemicals as plant toxins and insecticides in turn that is leading to increased resistance in insect populations. So far, most of them were isolated from dipteran and lepidopteran insect species and more specifically from pest or diseases vector species. So in insects, P450 genes can be assigned to one of four clades: CYP2, CYP3, CYP4 and the mitochondrial CYP clade. The CYP3 clande is subdivided again into the CYP6, CYP9, CYP28, CYP308-310, CYP317, CYP321, CYP324, CYP329, CYP332, CYP336-338 and CYP345-348 families. Indeed, Many CYP9 family members are known to participate in detoxification pathways associated with insecticide resistance. Hence, they are considered as key enzymes in order to visualize many metabolic pathways and based on these signals that enable to formulate desirable remedial actions [1,[10][11]. Having these in mind, this review aimed at P450s in silkworm species.

Cytochrome P450 Genes in Bombyx Mori
Based on the advances in silkworm genome projects, a new genome wide analysis of cytochrome P450s genes was performed by different scientists. Consequently, their finds revealed that Bombyx mori genome has not similar putative p450 gene numbers. That is Li and his colleagues were discovered that 86 putative P450s in silkworm genome, which are thought to belong to 32 subfamilies. Further, they mad a comparative genomic analysis with Drosophila melanogaster, then the result revealed that the two insects have some similar P450 distribution patterns but still have some obvious differences. Especially, the diverse distribution exists in 7 p450 subfamilies, which are CYP4A, CYP4C, CYP4D, CYP6A, CYP6AE, CYP6B and CYP9A [11]. But, Li and his colleagues in other study, they came up with different result of predication number of putative p450s which was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported [12]. On the contrary, Ai and his colleagues predicted that based on the same genome project and analysis of cytochrome P450 genes they obtained a total of 84 CYP-related sequences which could be classified into 26 families and 47 subfamilies according to standard nomenclature. According to their report out of total eighty four genes, seventy eight of them were appeared to be functional and six were probable pseudogenes. Furthermore, their result revealed that the distribution of Bombyx mori P450s in the genome depicted that most of them are tandem arranged on chromosomes, while merely 34 genes were present as singletons, with 8 clusters including 3 or more than 3 genes . On the other hand, they came up with B. mori and H. armigera CYP9A genes clustered in species-specific groups of three P450s, with the exception of the orthologous CYP9A22/CYP9A14 pair, which indicates that the gene expansions are independent species-specific events relative to the last common ancestor. As different phytophagous insects, B. mori and H. armigera encounter many predictably different chemistries and xenobiotics in their respective host plants and their distinct environments [3]. Hence, due to these dynamic and selective pressures on gene copies mechanisms exposed them to engage into a gradual modification which will create genes with specific functions than their precursors. In general, we infer that these gene evolution scenario will drive insect P450s to diversify rapidly.

Conclusion
Even though different studies were resulted in different number of p450 genes, the attempt made in identification of putative cytochrome p450 gene numbers and their expression profiles provides new insights into the similarities and differences in the structure, evolution, expression, and functions of genes, and raises many interesting questions which will need further scrutiny and the orthologous relationships among different insect CYP9A genes as well. Hence, it needs further investigation at least to arrive on specific number of cytochrome p450 genes in Bombyx mori to avoid ambiguities.


s2fieldsofstudy: Biology Chemistry

text:
The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function

It is increasingly recognized that synthesis and turnover of cardiac triglyceride (TG) play a pivotal role in the regulation of lipid metabolism and function of the heart. The last step in TG synthesis is catalyzed by diacylglycerol:acyltransferase (DGAT) which esterifies the diacylglycerol with a fatty acid. Mammalian heart has two DGAT isoforms, DGAT1 and DGAT2, yet their roles in cardiac metabolism and function remain poorly defined. Here, we show that inactivation of DGAT1 or DGAT2 in adult mouse heart results in a moderate suppression of TG synthesis and turnover. Partial inhibition of DGAT activity increases cardiac fatty acid oxidation without affecting PPARα signaling, myocardial energetics or contractile function. Moreover, coinhibition of DGAT1/2 in the heart abrogates TG turnover and protects the heart against high fat diet-induced lipid accumulation with no adverse effects on basal or dobutamine-stimulated cardiac function. Thus, the two DGAT isoforms in the heart have partially redundant function, and pharmacological inhibition of one DGAT isoform is well tolerated in adult hearts.

It is increasingly recognized that synthesis and turnover of cardiac triglyceride (TG) play a pivotal role in the regulation of lipid metabolism and function of the heart. The last step in TG synthesis is catalyzed by diacylglycerol:acyltransferase (DGAT) which esterifies the diacylglycerol with a fatty acid. Mammalian heart has two DGAT isoforms, DGAT1 and DGAT2, yet their roles in cardiac metabolism and function remain poorly defined. Here, we show that inactivation of DGAT1 or DGAT2 in adult mouse heart results in a moderate suppression of TG synthesis and turnover. Partial inhibition of DGAT activity increases cardiac fatty acid oxidation without affecting PPARα signaling, myocardial energetics or contractile function. Moreover, coinhibition of DGAT1/2 in the heart abrogates TG turnover and protects the heart against high fat diet-induced lipid accumulation with no adverse effects on basal or dobutaminestimulated cardiac function. Thus, the two DGAT isoforms in the heart have partially redundant function, and pharmacological inhibition of one DGAT isoform is well tolerated in adult hearts.
Cardiac triglyceride (TG) accumulation is a common clinical feature observed in older individuals and patients with diabetes 1,2 . Although increased cardiac TG content has previously been considered as a marker of lipotoxic cardiomyopathy, recent work showed that enhancing cardiac triglyceride production by diacylglycerol:acyltransferase 1 (DGAT1) overexpression was protective against lipotoxicity and ischemia-reperfusion injury 3,4 . These findings suggest that cardiac lipid turnover plays an important role in stress response. When cardiac TG lipolysis is blocked by deletion of adipose triglyceride lipase (ATGL) it results in a dramatic steatosis, cardiac dysfunction and early death 5 . Alternatively, increasing cardiac lipolysis by overexpression of ATGL protects against pressure overload and diabetic cardiomyopathy 6,7 . These data suggest that TG synthesis followed by breakdown, otherwise known as TG turnover, plays a pivotal role in cardiac physiology and diseases. Although studies targeting TG metabolism through ATGL or DGAT1 overexpression have been enlightening, few studies exist that have evaluated the effect of decreased TG production on cardiac metabolism and health. This is an important issue as inhibition of TG synthesis is being considered as a therapeutic measure for hyperlipidemia [8][9][10] .
The final step in TG synthesis is catalyzed by DGAT 1 and 2, both of which are present in the heart. The DGAT1-null mice were previously shown to be resistant to diet-induced obesity with no cardiac phenotype, mainly due to reduced lipid uptake in the intestine 9-12 . The DGAT1-null mice had normal levels of TG and diacylglycerol (DAG) in the heart 10 while cardiac specific deletion of DGAT1 was sufficient to increase DAG levels without effecting TG levels 13 . The in vivo rate of TG synthesis or turnover in the heart has never been assessed in these models. The relative contribution of the two DGAT isoforms in cardiac triglyceride metabolism has not been addressed either.
In addition to protection against lipotoxicity, TG turnover has been suggested to be critical for modulation of fatty acid oxidation via regulating the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα), possibly by contributing a fatty acid ligand. Previous studies have shown that either extracellular TG lipolysis by lipoprotein lipase (LPL) or intracellular TG lipolysis by ATGL can increase PPARα activity 5,14,15 . In rodent hearts, TG turnover rate was shown closely correlated with PPARα activity and cardiac function 5,14,15 . In failing human hearts, DGAT expression was shown to be decreased which was considered a pathogenic mechanism for decreased fatty acid oxidation and lipotoxicity 16 .
In the present study we determined the role of DGAT 1 and 2 in TG synthesis and turnover in the heart as well as their contributions to cardiac fatty acid metabolism. Utilizing an inducible cardiac-specific DGAT1 deletion Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, 98109, USA. Nathan D. Roe and Michal K. Handzlik contributed equally to this work. Correspondence and requests for materials should be addressed to R.T. (email: rongtian@uw.edu) mouse model (iKO) together with DGAT2-specific inhibitor, we were able to achieve graded inhibition of TG synthesis and turnover in adult mouse hearts. We observed that deletion of DGAT1 only modestly reduced TG synthesis from exogenous fatty acids which correlated with increased fatty acid oxidation and had no effect on cardiac function up to 1 year of age. Furthermore, we found that DGAT2 compensated for DGAT1 function in iKO and that inhibition of both isoforms abrogated TG synthesis but did not alter cardiac response to high fat diet.

Results
Deletion of DGAT1 has no effect on cardiac TG content and function. To investigate the role of DGAT1 in the heart, we generated cardiac-specific constitutive and inducible DGAT1 KO mouse models (cKO and iKO, respectively). Both models showed reduced DGAT1 mRNA (Fig. 1A) and protein ( Fig. 1B) with no effects on DGAT2 mRNA expression or cardiac TG content (Fig. 1A,D). As evidenced by electron microscopy, there were no differences between genotypes for cardiac lipid droplet number and morphology ( Fig. 1E-G). Relative to control mice, echocardiography analysis showed no differences in cardiac geometry or function of cKO and iKO mice for up to 12 months (Fig. 1H, Supplemental Table 2). To focus on the effects of DGAT inhibition in adult hearts, we used iKO model for the rest of the study.
Cardiac TG synthesis is modestly reduced with loss of DGAT1. Using 13 C NMR spectroscopy we examined fatty acid incorporation rate into the TG pool of isolated hearts perfused with 13 C labeled substrates. The iKO showed a 30% lower rate of TG synthesis from exogenous fatty acids than the control ( Fig. 2A). GC-MS analysis of cardiac triglycerides extracted from the heart at the end of labeling confirmed reduction in 13 C labeled fatty acids in the TG pool (Fig. 2B). Moreover, total cardiac TG content was unaffected by inducible DGAT1 deletion (Fig. 2C). Collectively, these data show a modest decrease in cardiac TG turnover following DGAT1 deletion in adult hearts.

Increased oxidation of exogenous fatty acids in DGAT1 iKO hearts.
To assess the effects of reduced TG turnover on substrate oxidation, the relative contribution of 13 C labeled fatty acids and glucose along with unlabeled substrates to the tricarboxylic acid (TCA) cycle was assessed by 13 C-NMR techniques. Compared to control, increased contribution from exogenous fatty acid and reduced glucose contribution to total oxidative metabolism was observed in iKO hearts (Fig. 3A). Moreover, there was a negative correlation between fatty acid oxidation and the incorporation of exogenous fatty acids into the TG pool (Fig. 3B), suggesting that decreased TG synthesis is associated with increased oxidation of exogenous fatty acids. These changes in fatty acid metabolism did not affect cardiac function or energetics in the perfused iKO hearts (Fig. 3C,D). Additionally, these hearts responded normally to increased workload in vivo induced by dobutamine (Fig. S1). These data suggest that adult hearts are able to adapt to a moderate decrease in TG turnover caused by DGAT1 deletion and are able to maintain cardiac energetics or function.

DGAT1 iKO hearts respond normally to high fat diet.
To evaluate the effect of reduced TG synthesis on lipid loading-induced metabolic remodeling, we subjected control and iKO mice to a high fat diet (HFD, 60% kcal from fat) for 5 weeks. Body weight gains were similar in control and iKO mice after the 5 week HFD ( Fig. S2A-B). Cardiac TG or DAG content did not change in control or iKO hearts after 5 weeks of a high fat diet (Fig. 4A,B). However, the 5-week HFD was sufficient to stimulate PPARα activity and increase fatty acid oxidation as previously described 17 . Interestingly, increases in PPARα activity and fatty acid oxidation were similar in iKO and control hearts (Fig. 4C,D) suggesting that the modest reduction in triglyceride turnover did not affect hearts' ability to activate PPARα and increase fatty acid oxidation in response to increased lipid load.

Role of DGAT2 in cardiac TG metabolism.
Considering relatively mild effects of DGAT1 deficiency on cardiac lipid metabolism, we next sought to explore the relative contribution of the two DGAT isoforms in the heart. Quantitative measurements of the mRNA levels showed greater abundance of DGAT2 mRNA in the heart compared to DGAT1 (~1 vs. ~0.25 pmol/mg RNA respectively, Fig. 5A). To evaluate the contribution of DGAT2 to TG synthesis, we perfused hearts from control or iKO mice with DGAT2 inhibitor (5 µM) 18 . The DGAT2 inhibitor alone has very modest effect but inhibition of both isoforms substantially reduced 13 C fatty acid incorporation into TG pool in the heart as measured by NMR spectroscopy and GCMS (Fig. 5B,C). Moreover, perfusion with DGAT2 inhibitor for 60 minutes did not alter cardiac TG levels in control or iKO hearts (Fig. S3A) suggesting that inhibition of both DGAT isoforms effectively abrogated TG turnover in the heart. Furthermore, we found that inhibition of TG turnover resulted in a rapid downregulation of PPARα target gene expression with no change in cardiac function ( Fig. 5D and Fig. S3B), and DGAT2 inhibitor did not further change substrate oxidation in iKO during the perfusion period (Fig. S3C).

Cardiac effects of DGAT1/2 inhibition in vivo.
To explore how DGAT1/2 coinhibition modulates in vivo cardiac lipid metabolism, we treated iKO mice with DGAT2 inhibitor for 5 days. Under fasting and fed conditions, both control and iKO mice treated with DGAT2 inhibitor had lower cardiac triglyceride content compared to vehicle treated controls (Fig. 6A). To investigate whether prolonged DGAT1/2 coinhibition modifies cardiac lipid metabolism and function during sustained lipid overload we subjected mice to 5-week HFD. Treatment with DGAT2 inhibitor in mice on control diet showed a modest effect on body weight with no significant change of blood TG level. In HFD-fed mice, however, DGAT2 inhibitor reduced body weight gain and protected against the increase of blood TG level (Fig. 6B,C). Cardiac TG was also lower in iKO mice treated with DGAT2 inhibitor (Fig. 6D). The downregulation of cardiac TG level did not affect cardiac PPARα signaling or its response to HFD (Fig. 6E). Interestingly, while basal cardiac function was unaltered between genotypes on control diet or HFD, dobutamine-induced increase in ejection fraction was attenuated in mice subjected to the HFD which was not altered by DGAT inhibition (Fig. 6F).

Discussion
In this study we show that both isoforms of DGAT contribute to triglyceride synthesis in the heart with partially redundant function. Loss of DGAT1 in the adult mouse heart exerts a moderate inhibition on cardiac triglyceride synthesis accompanied by increased oxidation of exogenous fatty acids. Inhibition of both enzymes abrogates triglyceride synthesis and turnover in the perfused heart leading to the downregulation of PPARα target gene expression. Treatment with DGAT2 inhibitor in mice with cardiac-specific DGAT1 deficiency prevents TG accumulation in the heart during 5-week high fat diet. It however, does not change the PPARα signaling or cardiac performance under control or high fat diet conditions. It has been well recognized that myocardial TG pool is highly dynamic although the heart has very limited capacity for lipogenesis. The exact role of the TG turnover is not entirely clear but several potential functions have been proposed including preventing the generation of lipotoxic species during lipid overload, and supply fuel for oxidation upon energy stress. Several recent studies have suggested that TG lipolysis may regulate fatty acid oxidation by supplying ligands for PPARα activation 5 . Critical evidence in support of this notion was derived from mice with ATGL deletion in which blockade of TG lipolysis resulted in decreased fatty acid oxidation that could be rescued by pharmacological activation of PPARα. However, blockade of TG lipolysis also causes substantial steatosis that confounds the ATGL deletion phenotype. In this study, we find that TG turnover can be inhibited by targeting TG synthesis with no change of TG pool size. Using this approach, we show that partial inhibition of TG turnover by deleting DGAT1 in adult mouse heart does not impair PPARα transcriptional activity. The heart adapts to the decreased TG synthesis by increasing oxidation of the exogenous fatty acids without changes in lipid metabolism homeostasis or myocardial energetics. In response to high fat diet, not only does DGAT1 deficiency not affect cardiac function, but the iKO heart retains its ability to upregulate PPARα activity and remodel metabolism similar to that of the WT. This is unexpected given the growing body of evidence suggesting a close association of TG metabolism and PPARα activity with either TG turnover regulating PPARα activity 5,15,19 , or PPARα activity itself increasing TG turnover 20 . Furthermore, contrary to the previous studies suggesting that reduced TG synthesis and turnover would increase lipotoxic species in the heart 3,13,21 , DAG content in the iKO hearts did not change at baseline or following a high fat diet. These observations have led us to conclude that inhibition of DGAT1 in the adult heart exert insufficient effect on TG synthesis rates to impair cardiac function, lipid homeostasis, or PPARα activation.
Previous studies have revealed important differences in structure and subcellular localization of the two DGAT enzymes, suggesting that DGAT1 and 2 participate in different pathways of TG synthesis 22,23 . A model based on this premise proposes that DGAT1 is primarily involved in esterifying exogenous fatty acids to replenish the TG pool while DGAT2 is responsible for incorporating endogenously synthesized fatty acids into TG 22 . Since the heart is not a lipogenic organ, DGAT1 would be expected to play a primary role in TG synthesis. Interestingly, DGAT2 is highly expressed in the heart but its function has not been assessed due to neonatal lethality of DGAT2 knockout mice 24 . Using dynamic 13 C-NMR technique, here we show that the two enzymes have partially redundant function in the heart; inhibition of either enzyme only modestly affects the rate of fatty acids incorporation into the TG pool. Inhibition of both DGAT activities results in near complete loss of TG synthesis in the heart. This is consistent with the profound reduction of TG in fetal mice lacking both enzymes 25 , thus, supporting the notion that these two DGATs are jointly responsible for TG synthesis in the heart. Compensation by DGAT2 is likely an adaptive mechanism for DGAT1 deficiency in the heart. Although deletion of DGAT1 in the adult mouse hearts does not trigger any increases of DGAT2 mRNA level, we are unable to determine the protein level or post-translational modification of DGAT2 in the iKO hearts due to the lack of DGAT2-specific antibody. Recent studies have shown that ubiqutination or translocation of DGAT2 could change its stability or function in non-cardiac cells 26 , whether a similar mechanism contributes to cardiac triglyceride synthesis warrants further investigation.
Targeting TG synthesis has been proposed as a therapeutic strategy for hyperlipidemia for more than a decade 9 . Multiple small molecular inhibitors of DGAT, especially DGAT2, have been developed recently 18,27,28 . However, previous studies have suggested that inhibition of TG synthesis and turnover in the heart could contribute to lipotoxicity and hence cautioned the use of pharmacological inhibition of DGAT. In contrast, we find that inhibition of one isoform of DGAT in the adult heart is well tolerated. Once a near complete inhibition of TG synthesis and turnover is achieved by perfusing iKO hearts with DGAT2 inhibitor, expression of PPARα target genes is significantly downregulated. Inhibition of both DGAT isoforms in vivo, although effectively reduces TG content in the heart, do not affect PPARα activity or cardiac function under normal or high fat diet condition. We speculate that pharmacological inhibition of DGAT2 in the heart is only intermittent in vivo, thus resulting in a state of partial suppression of TG turnover despite the reduction of TG level. Collectively, these observations suggest that a low rate of TG turnover is sufficient to maintain PPARα activity, and furthermore, adult hearts can adapt to partial inhibition of DGAT activity. It remains to be determined whether this degree of DGAT inhibition affects cardiac responses to other chronic stresses such as pressure overload.

Conclusions
In the present study, we find that both isoforms of DGAT contribute to cardiac TG synthesis with partial redundancy. Coinhibition of DGAT1 and 2 in the heart is necessary to abrogate cardiac TG turnover. Moreover, inhibiting DGAT1 and 2 in the heart during chronic, sustained lipid loaded protects against cardiac lipid accumulation without significant changes of cardiac function.   13 C labeling of TG pool in the control and iKO hearts perfused with DGAT2 inhibitor (B), mass spectrometry analysis of 13 C labeling of TG pool in control and iKO hearts perfused with DGAT2 inhibitor (C) and PPARα signaling between WT and iKO hearts perfused with 5 µM DGAT2 inhibitor (D). Data are presented as mean ± SEM (n = 3-7). *P < 0.05 vs. control Figure 6. Coinhibition of DGAT1 and DGAT2 during high fat diet (HFD). Cardiac TG level was decreased with DGAT2 inhibition or with iKO + DGAT2 inhibition under fed and fasting conditions (A). Coinhibition of DGAT1 and DGAT2 attenuated body weight gain (B) and reduced serum (C) and cardiac (D) TG content following HFD. PPARα signaling (E) and basal cardiac function (F) were unaffected in iKO + DGAT2 mice on a HFD compared to control. Dobutamine-induced increase in cardiac function was attenuated by HFD with no differences between genotypes (F). Data are presented as mean ± SEM (n = 4-8). *P < 0.05, **P < 0.01, ***P < 0.001. † P < 0.05 vs. corresponding control group, § P < 0.05 mean effect of diet. Data analyzed by oneway ANOVA (A) and two-way ANOVA (B-F). transgenic mice to generate cardiac-specific DGAT1 knockout (cKO). Age and sex-matched littermate DGAT1 flox/flox mice were used as controls. For iKO mice, tamoxifen (20 mg/kg/d, i.p.) was given for 5 days to 8-week old MCM-DGAT1 flox/flox mice and mice were analyzed 4 weeks after final tamoxifen injection. For high fat diet treatment, 3-month old mice were fed a diet with 60.3% kcal from fat (HFD, Research Diets TD.09766) or a control diet with 16.8% kcal from fat (CD, Research Diets TD.120455) for 5 weeks. For DGAT2 inhibitor treatment, control or iKO mice 4 weeks after tamoxifen treatment were orally gavaged (30 mg/kg, BID) of the DGAT2 inhibitor PF-06424439-27 in 0.5% methylcellulose for 5 days and tissues harvested with or without overnight fasting. Separate cohort of mice on HFD or CD were treated with the same dose of PF-06424439-27 for 5 weeks. All mice were kept on a 12 h light/dark cycle with ad libitum access to food and water.
Echocardiography. Transthoracic echocardiography was collected from mice anesthetized with isoflurane (1-2%) using a Vevo 2100 (VisualSonics) in short axis mode. Dobutamine stress echo was conducted by administration of dobutamine (3 mg/kg, i.p.) immediately after the baseline function assessment and repeating the echocardiographic measurements 10 minutes after dobutamine injection. Data for each mouse is an average of 3 cardiac cycles taken at comparable heart rates.
Mice were heparinized (100 U/mouse), anesthetized with pentobarbital (190 mg/kg, I.P.), hearts excised and perfused at 80 mmHg perfusion pressure at 37 °C with a KH buffer containing the following in mM: 0.5 EDTA, 5.3 KCl, 1.2 MgSO4, 118 NaCl, 25 NaHCO3, 2 CaCl2, 0.4 mixed long chain fatty acids, 5.5 glucose, 1.2 lactate and 50 µU/mL insulin. Ex vivo cardiac phosphocreatine (PCr) and ATP contents were determined using 31 P NMR as previously described 4 . Contribution of individual substrates to total oxidative metabolism was determined using 13 C NMR 4 . Specifically, hearts perfused with 1,6-13 C glucose and U-13 C mixed fatty acids were freeze-clamped following 40 min labelling perfusion, tissues were powdered and metabolites extracted with perchloric acid. Proton-decoupled 13 C NMR spectra of tissue extracts were acquired on a 14 T magnet using TopSpin software (Bruker 600; Billerica, MA) and contributions of labeled (glucose and fatty acids) and unlabeled (exogenous lactate and endogenous glycogen and TG) substrates to total oxidative metabolism were determined by modeling of citric acid cycle flux using the glutamate C3 and C4 isotopomer peak areas (tcaCALC, Dallas, TX).

TG synthesis.
Incorporation of 13 C-labeled mixed fatty acids into the TAG pool was analyzed by monitoring the resonance of the methylene carbon at ~31 ppm via 13 C NMR spectroscopy as previously described 4 . Proton-decoupled carbon spectra were determined on isolated hearts perfused with 13 C-labeled mixed fatty acids over a 60-min period. Peak areas of the methylene resonance of each sequential spectrum were quantified using software from Advanced Chemistry Development Laboratories (ACD Laboratories, Toronto, Ontario, Canada) and corrected for 13 C natural abundance.
Electron Microscopic Analysis. Thin heart sections were cut and rinsed in a KH buffer containing 20 mM KCl to arrest the muscle in diastole. Sections were immediately placed into a fixation solution containing 0.4% glutaraldehyde in 0.1 M sodium cacodylate buffer. Fixed tissue samples were processed and images taken using a Transmission Electron Microscope (JEOL1230). Lipid droplet number and morphology were quantified using ImageJ analysis software. Data for each heart were calculated as average value of 40 random images.
Biochemical Assays. Total lipids were extracted using the Folch extraction procedure and quantified using the sigma colorimetric assay kit as previously described 31 . Briefly, heart tissues were weighed and homogenized in 2:1 chloroform:methanol, incubated on ice for 30 min, then centrifuged at 3000 rpm for 12 minutes. The top layer was discarded, the bottom layer was dried under N2 gas and reconstituted in 1% Triton X-100:Isopropanol. Individual lipid classes were extracted as described above were loaded onto an NH2 column (Agilent BondElut) preconditioned with hexane and neutral lipids were extracted with 2:1 Chlorofom:Isopropanol, dried under N2 gas, reconstituted in hexane and loaded onto another preconditioned NH2 column 4,32 . TG were eluted from the column by the addition of 1% diethyl ether:10% methylene chloride in hexane and DAG with 15% ethyl acetate in hexane dried under N2 gas. DAG was quantified using the triglyceride assay kit with a DAG standard curve. Fatty acid composition and 13 C labeling patterns was assessed using a gas chromatograph mass spectrometer (GCMS, Shimadzu QP2010) with a 0.25 mm inner diameter column (Restek Rtx-225) on fatty acid methyl esthers (FAMES) made from the triglyceride fraction obtained using solid phase separation as previously described 33 . Real Time PCR. RNA from frozen heart tissue was isolated using the RNeasy fibrous extraction kit (Qiagen) and cDNA was synthesized using the Omniscript (Qiagen) reverse transcriptase according to the manufacturer's recommendations. PCR reactions containing equal cDNA concentrations were set up in duplicate using Sybr green (BioRad) on a rotogene using concentrated cDNA as a standard. All values were normalized to 18 S concentration and expressed as a fold change from control. Primer sequences for all genes are described in Supplemental Table 1. Quantitative measurements of DGAT1 and DGAT2 levels in mouse heart tissue were performed based on standard curves generated using full-length amplicons supplied by Integrated DNA technologies.
Western blotting. Whole heart proteins were extracted from flash frozen hearts using 10-20 mg of wet weight tissue using RIPA buffer containing 150 mM NaCl, 50 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1 mM EDTA, pH 7.4. Briefly, frozen hearts were homogenized in RIPA buffer using bullet blender and placed on ice for 30 min. Next, samples were centrifuged for 10 min at 4 °C at 20,000 g. Supernatant was collected and its protein content determined using BCA assay kit (BCA protein assay kit, Pierce Biotechnology, USA). Protein samples were run a 10% Bis-Tris acrylamide gel for 2 h at constant 100 V and transferred on a polyvinylidenedifluoride membrane (PVDF) for 2 h at constant 250 mA in an ice-chilled transfer SCientifiC RepoRTs | (2018) 8:4983 | DOI:10.1038/s41598-018-23223-7 tank. The membrane was blocked and incubated overnight at 4 °C with mouse anti-DGAT1 antibody (Santa Cruz, #sc-271934) and rabbit anti-GAPDH antibody (Cell Signaling, #D16H11). Membranes were then washed and incubated with corresponding anti-mouse or goat anti-rabbit HRP-conjugated secondary antibody (R&D Systems). After washing membranes were incubated with enhanced chemiluminesence (ECL) detection solution (Supersignal Westpico Chemiluminescent Substrate, Thermo, #34080) and exposed to X-ray film (HyBlot CL, Denville Scientific, Inc., NJ, US). Intensity was measured by ImageJ densitometry software.
Statistics. All data are presented as mean ± SEM. Statistical analysis was conducted using a student's t-test for experiments with 2 groups, one-way ANOVA was used for experiments of three or more groups. Two-way ANOVA was used for experiments with repeated measures as specified in the figure legend. Graphs and statistical analyses were performed using Graph Pad Prism 6.0. Statistical significance was set at P < 0.05. Data availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).