Search

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops 1,2 . Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes 3-5 . Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type 6-9 . Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria 10-12 , we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes., (© 2024. The Author(s).)

Importance: Early-onset sepsis (EOS) remains a potentially fatal newborn condition. Ongoing surveillance is critical to optimize prevention and treatment strategies., Objective: To describe the current incidence, microbiology, morbidity, and mortality of EOS among a cohort of term and preterm infants., Design, Setting, and Participants: This prospective surveillance study included a cohort of infants born at a gestational age (GA) of at least 22 weeks and birth weight of greater than 400 g from 18 centers of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network from April 1, 2015, to March 31, 2017. Data were analyzed from June 14, 2019, to January 28, 2020., Main Outcomes and Measures: Early-onset sepsis defined by isolation of pathogenic species from blood or cerebrospinal fluid culture within 72 hours of birth and antibiotic treatment for at least 5 days or until death., Results: A total of 235 EOS cases (127 male [54.0%]) were identified among 217 480 newborns (1.08 [95% CI, 0.95-1.23] cases per 1000 live births). Incidence varied significantly by GA and was highest among infants with a GA of 22 to 28 weeks (18.47 [95% CI, 14.57-23.38] cases per 1000). No significant differences in EOS incidence were observed by sex, race, or ethnicity. The most frequent pathogens were Escherichia coli (86 [36.6%]) and group B streptococcus (GBS; 71 [30.2%]). E coli disease primarily occurred among preterm infants (68 of 131 [51.9%]); GBS disease primarily occurred among term infants (54 of 104 [51.9%]), with 24 of 45 GBS cases (53.3%) seen in infants born to mothers with negative GBS screening test results. Intrapartum antibiotics were administered to 162 mothers (68.9%; 110 of 131 [84.0%] preterm and 52 of 104 [50.0%] term), most commonly for suspected chorioamnionitis. Neonatal empirical antibiotic treatment most frequently included ampicillin and gentamicin. All GBS isolates were tested, but only 18 of 81 (22.2%) E coli isolates tested were susceptible to ampicillin; 6 of 77 E coli isolates (7.8%) were resistant to both ampicillin and gentamicin. Nearly all newborns with EOS (220 of 235 [93.6%]) displayed signs of illness within 72 hours of birth. Death occurred in 38 of 131 infected infants with GA of less than 37 weeks (29.0%); no term infants died. Compared with earlier surveillance (2006-2009), the rate of E coli infection increased among very low-birth-weight (401-1500 g) infants (8.68 [95% CI, 6.50-11.60] vs 5.07 [95% CI, 3.93-6.53] per 1000 live births; P = .008)., Conclusions and Relevance: In this study, EOS incidence and associated mortality disproportionately occurred in preterm infants. Contemporary cases have demonstrated the limitations of current GBS prevention strategies. The increase in E coli infections among very low-birth-weight infants warrants continued study. Ampicillin and gentamicin remained effective antibiotics in most cases, but ongoing surveillance should monitor antibiotic susceptibilities of EOS pathogens.

Importance: Racial/ethnic disparities in quality of care among extremely preterm infants are associated with adverse outcomes., Objective: To assess whether racial/ethnic disparities in major outcomes and key care practices were changing over time among extremely preterm infants., Design, Setting, and Participants: This observational cohort study used prospectively collected data from 25 US academic medical centers. Participants included 20 092 infants of 22 to 27 weeks' gestation with a birth weight of 401 to 1500 g born at centers participating in the National Institute of Child Health and Human Development Neonatal Research Network from 2002 to 2016. Of these infants, 9316 born from 2006 to 2014 were eligible for follow-up at 18 to 26 months' postmenstrual age (excluding 5871 infants born before 2006, 2594 infants born after 2014, and 2311 ineligible infants including 64 with birth weight >1000 g and 2247 infants with gestational age >26 6/7 weeks), of whom 745 (8.0%) did not have known follow-up outcomes at 18 to 26 months., Main Outcomes and Measures: Rates of mortality, major morbidities, and care practice use over time were evaluated using models adjusted for baseline characteristics, center, and birth year. Data analyses were conducted from 2018 to 2019., Results: In total, 20 092 infants with a mean (SD) gestational age of 25.1 (1.5) weeks met the inclusion criteria and were available for the primary outcome: 8331 (41.5%) black infants, 3701 (18.4%) Hispanic infants, and 8060 (40.1%) white infants. Hospital mortality decreased over time in all groups. The rate of improvement in hospital mortality over time did not differ among black and Hispanic infants compared with white infants (black infants went from 35% to 24%, Hispanic infants went from 32% to 27%, and white infants went from 30% to 22%; P = .59 for race × year interaction). The rates of late-onset sepsis among black infants (went from 37% to 24%) and Hispanic infants (went from 45% to 23%) were initially higher than for white infants (went from 36% to 25%) but decreased more rapidly and converged during the most recent years (P = .02 for race × year interaction). Changes in rates of other major morbidities did not differ by race/ethnicity. Death before follow-up decreased over time (from 2006 to 2014: black infants, 14%; Hispanic infants, 39%, white infants, 15%), but moderate-severe neurodevelopmental impairment increased over time in all racial/ethnic groups (increase from 2006 to 2014: black infants, 70%; Hispanic infants, 123%; white infants, 130%). Rates of antenatal corticosteroid exposure (black infants went from 72% to 90%, Hispanic infants went from 73% to 83%, and white infants went from 86% to 90%; P = .01 for race × year interaction) and of cesarean delivery (black infants went from 45% to 59%, Hispanic infants went from 49% to 59%, and white infants went from 62% to 63%; P = .03 for race × year interaction) were initially lower among black and Hispanic infants compared with white infants, but these differences decreased over time., Conclusions and Relevance: Among extremely preterm infants, improvements in adjusted rates of mortality and most major morbidities did not differ by race/ethnicity, but rates of neurodevelopmental impairment increased in all groups. There were narrowing racial/ethnic disparities in important care practices, including the use of antenatal corticosteroids and cesarean delivery.

Objective: Little is known about how very low birth weight (VLBW) affects survival and morbidities among infants with trisomy 13 (T13) or trisomy 18 (T18). We examined the care plans for VLBW infants with T13 or T18 and compared their risks of mortality and neonatal morbidities with VLBW infants with trisomy 21 and VLBW infants without birth defects., Methods: Infants with birth weight 401 to 1500 g born or cared for at a participating center of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network during the period 1994-2009 were studied. Poisson regression models were used to examine risk of death and neonatal morbidities among infants with T13 or T18., Results: Of 52,262 VLBW infants, 38 (0.07%) had T13 and 128 (0.24%) had T18. Intensity of care in the delivery room varied depending on whether the trisomy was diagnosed before or after birth. The plan for subsequent care for the majority of the infants was to withdraw care or to provide comfort care. Eleven percent of infants with T13 and 9% of infants with T18 survived to hospital discharge. Survivors with T13 or T18 had significantly increased risk of patent ductus arteriosus and respiratory distress syndrome compared with infants without birth defects. No infant with T13 or T18 developed necrotizing enterocolitis., Conclusions: In this cohort of liveborn VLBW infants with T13 or T18, the timing of trisomy diagnosis affected the plan for care, survival was poor, and death usually occurred early.

Ingestion of Toxoplasma gondii tissue cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of meat and meat products to eliminate viable T. gondii tissue cysts would provide a means to protect consumers. In this study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.

Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of C. parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study examined the efficacy of two alternative food-processing treatments, e-beam irradiation and microwave energy, on the viability of C. parvum oocysts in Eastern Oysters (Crassostrea virginica), which were artificially infected with the Beltsville strain of C. parvum. The effects of the treatments were evaluated by oral feeding of the processed oyster tissues to neonatal mice. Significant reductions (P<0.05) in infectivity were observed for in-shell and shucked oysters treated with e-beam irradiation at doses of 1.0, 1.5, or 2 kGy vs. untreated controls. A dose of 2 kGy completely eliminated C. parvum infectivity and did not adversely affect the visual appearance of the oysters. Oyster tissue treated with microwave exposures of 1 s (43.2 degrees C), 2 s (54.0 degrees C), and 3 s (62.5 degrees C) showed a reduction in C. parvum mouse infectivity, but the effects were not significantly different (P>0.05) from controls. Microwave energy treatments at 2 and 3 s showed extensive changes in oyster meat texture and color. Thus, because of lack of efficacy and unacceptable tissue changes, microwave treatment of oysters is not considered a viable food-processing method.

Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of Cryptosporidium parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study identified the efficacy of a non-thermal alternative food-processing treatment, high hydrostatic pressure processing (HPP), on the viability of C. parvum oocysts in the Eastern oysters Crassostrea virginica. Oysters were artificially exposed to 2 x 10(7) oocysts of the Beltsville strain of C. parvum in seawater and subjected to HPP treatments. The effects of the treatments were evaluated by inoculation of the processed oyster tissues into neonatal mice. High-pressure processing of shucked Eastern oysters at all pressures tested (305, 370, 400, 480, and 550 MPa) was significantly effective (P<0.05) in reducing the numbers of positive mouse pups fed treated oyster tissues exposed to C. parvum oocysts. A dose of 550 MPa at 180 s (s) of holding time produced the maximum decrease in numbers of C. parvum positive mouse pups (93.3%). Measurement of tristimulus color values of HPP-treated raw oysters at extended processing times from 120 s to 360 s at 550 MPa showed a small increase in whiteness of oyster meat. This non-thermal processing treatment shows promise for commercial applications to improve safety of seafood and reduce public health risks from cryptosporidiosis.

High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for 1 min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were rendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.

We have been collaborating since 1992 in studies on southern sea otters (Enhydra lutris nereis) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otters. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 15 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22-24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.