Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium ...parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.
Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of
around the world threatens the progress made against this disease. ...Therefore, identifying druggable and essential pathways in
parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity. Graphic abstract: Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.
Despite being politicized in its early stages, the Red Wolf (Canis rufus) Recovery Program (RWRP) existed for 30 years in eastern North Carolina with little attention paid to finding common ground ...between policy contestants. We situated our data from 62 key informant interviews within Stone's (2002) policy goals framework to evaluate red wolf recovery in eastern North Carolina, United States. Four goals underpin U.S. political culture and offer common ground with which to begin negotiation among policy contestants. Our results highlight challenges to achieving one definition prescribing how to treat each policy goal. The future of recovery within the red wolf's historic range could hinge on decision-makers' willingness to prioritize policy design over policy tool evaluation, developing a process by which to achieve an agreed-upon set of values underscoring red wolf recovery. This process must reconcile disagreements about what is fair or equitable treatment of landowners and include local-level experiences.
Populations of American Alligators (Alligator mississippiensis) generally are considered more abundant at present than historically; however, little information exists to assess the population of ...alligators in North Carolina at the northern extent of the species' range. Investigation of the factors influencing the distribution and abundance of alligators in North Carolina could shed light on the species' response to rapid environmental change in the region. We conducted a two-phase study: 1) to assess the distribution of alligators in North Carolina using a site-occupancy design; and 2) to assess the patterns in abundance using a repeated sampling design for population estimation. Results showed that both occupancy and abundance decreased in more northern sites, in sites with higher salinity, and in sites that were generally more westward. Sites sampled later in June were more likely to be occupied than those sampled earlier in the month. Abundance also increased with greater shoreline vegetation complexity and varied between lakes, rivers, and estuaries. Compared with studies from 30 years prior, the population seems fairly stable in terms of abundance and distribution. Given the northern limits of the species and the negative association with salinity, continued monitoring is warranted to understand changes in distribution and abundance with respect to predicted rates of sea-level rise, salinization, and urbanization locally around coastal cities like Wilmington.
The recent co-occurrence of red wolves (Canis rufus) and coyotes (Canis latrans) in eastern North Carolina provides a unique opportunity to study prey partitioning by sympatric canids. We collected ...scats from this region and examined them for prey contents. We used fecal DNA analysis to identify which taxa deposited each scat and multinomial modeling designed for mark–recapture data to investigate diets of sympatric red wolves and coyotes. Diets of red wolves and coyotes did not differ, but the proportion of small rodents in the composite scats of both canids was greater in the spring than in the summer. White-tailed deer (Odocoileus virginianus), rabbits (Sylvilagus spp.), and small rodents were the most common diet items in canid scats. The similarity of diet between red wolves and coyotes suggests that these 2 species may be affecting prey populations similarly.
Export of parasite proteins into the host erythrocyte is essential for survival of
during its asexual life cycle. While several studies described key factors within the parasite that are involved in ...protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by
in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70,
Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most
species and is found only in
and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in
to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the
ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of
to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor
erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of
.
Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of
spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria,
. An outstanding question in the field is how
undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the
species causing human disease, the chaperone PfHsp70x is unique to
, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.
When hybridizing species come into contact, understanding the processes that regulate their interactions can help predict the future outcome of the system. This is especially relevant in conservation ...situations where human activities can influence hybridization dynamics. We investigated a developing hybrid zone between red wolves and coyotes in North Carolina, USA to elucidate patterns of hybridization in a system heavily managed for preservation of the red wolf genome. Using noninvasive genetic sampling of scat, we surveyed a 2880 km2 region adjacent to the Red Wolf Experimental Population Area (RWEPA). We combined microsatellite genotypes collected from this survey with those from companion studies conducted both within and outside the RWEPA to describe the gradient of red wolf ancestry. A total of 311 individuals were genotyped at 17 loci and red wolf ancestry decreased along an east–west gradient across the RWEPA. No red wolves were found outside the RWEPA, yet half of individuals found within this area were coyotes. Hybrids composed only 4% of individuals within this landscape despite co‐occurrence of the two species throughout the RWEPA. The low proportion of hybrids suggests that a combination of active management and natural isolating mechanisms may be limiting intermixing within this hybrid system.
Painted Buntings (Passerina ciris) have been declining in the southeastern United States since the 1970s. A recent demographic assessment highlighted the importance of estimating demographic ...parameters, which have received little attention to date. The dearth of information is troublesome because attempts to reverse declining trends require a better understanding of the relationship between habitat quality and age- and sex-specific survival and recruitment rates. We used capture–mark–recapture data collected from 2007 to 2015 on Bald Head Island (BHI) and at Hammocks Beach State Park (HBSP) in North Carolina, USA, to estimate local age- and sex-specific annual survival rates and local population size and recruitment rates using programs MARK and LOLASURVIV. Juveniles had lower local survival rates than adults (HBSP: 0.28 ± 0.14 vs. 0.67 ± 0.06; BHI: 0.28 ± 0.04 vs. 0.57 ± 0.02). Local annual survival rates for males on BHI (0.50 ± 0.03) were lower than those for females (0.57 ± 0.02). Age-specific differences were consistent with known differential age-dependent survival skills, and sex-specific differences were consistent with the potential influence of sexual dichromism. Conservative estimates of population size on BHI averaged 101 juveniles and 263 adults annually. Annual in situ reproductive recruitment averaged 28 individuals plus an additional 120 new immigrants, indicating successful reproduction and connectivity with neighboring coastal populations. Local adult survival estimates from our 2 North Carolinian study populations were similar to high-end estimates from across the eastern and western range of the species (∼0.60). Finite observed population growth rate estimates between the BHI population (λ = 1.10) and a South Carolinian population (λ = 0.87) underscore the potential role of differential habitat quality and the importance of information from multiple sites, including nonbreeding grounds, for proper inferences about the status of the species. Reported vital rates provide a stronger foundation on which to base habitat quality as assessed with demographic parameters and to guide Painted Bunting conservation regionally.
Forage and nesting cover available to female Meleagris gallopavo (Wild Turkey) prior to nesting can influence nest success. Prescribed burns commonly are conducted during the dormant season in ...southern Pinus (pine) forests in part to improve vegetation conditions for prenesting Wild Turkeys and reduce risk of fire-related nest failure associated with growing-season burning. However, prescribed burning during the early growing season may provide beneficial food and cover for Wild Turkeys. Therefore, we investigated the influence of fire season and frequency and vegetation characteristics on female Wild Turkey habitat selection during prenesting in a Pinus palustris (Longleaf Pine) community managed with frequent growing-season prescribed fire in North Carolina. Growing-season fire history was not predictive of prenesting habitat selection. Females selected forest stands burned during the preceding dormant season, edges of non-forested cover, and creek drainages. On our study area, ericaceous shrubs along creek drainages provided nesting cover, and greater probability of use near creeks likely reflected females searching for potential nest sites. Recent dormant-season burns may provide an important source of nutrition for pre-nesting females and should be used in addition to growing-season burns when managing for Wild Turkeys.
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