Bioactive metabolites of OMEGA-6 and OMEGA-3 fatty acids are associated with inflammatory cytokine concentrations in maternal and infant plasma at the time of delivery.

BACKGROUND & AIMS: Inflammation is necessary for a healthy pregnancy. However, unregulated or excessive inflammation during pregnancy is associated with severe maternal and infant morbidities, such as pre-eclampsia, abnormal infant neurodevelopment, or preterm birth. Inflammation is regulated in part by the bioactive metabolites of omega-6 (n-6) and omega-3 (n-3) fatty acids (FAs). N-6 FAs have been shown to promote pro-inflammatory cytokine environments in adults, while n-3 FAs have been shown to contribute to the resolution of inflammation; however, how these metabolites affect maternal and infant inflammation is still uncertain. The objective of this study was to predict the influence of n-6 and n-3 FA metabolites on inflammatory biomarkers in maternal and umbilical cord plasma at the time of delivery. METHODS: Inflammatory biomarkers (IL-1ß, IL-2, IL-6, IL-8, IL-10, and TNFα) for maternal and umbilical cord plasma samples in 39 maternal-infant dyads were analyzed via multi-analyte bead array. Metabolites of n-6 FAs (arachidonic acid and linoleic acid) and n-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) were assayed via liquid chromatography-mass spectrometry. Linear regression models assessed relationships between maternal and infant inflammatory markers and metabolite plasma concentrations. RESULTS: Increased plasma concentrations of maternal n-6 metabolites were predictive of elevated pro-inflammatory cytokine concentrations in mothers; similarly, higher plasma concentrations of umbilical cord n-6 FA metabolites were predictive of elevated pro-inflammatory cytokine concentrations in infants. Higher plasma concentrations of maternal n-6 FA metabolites were also predictive of elevated pro-inflammatory cytokines in infants, suggesting that maternal n-6 FA status has an intergenerational impact on the inflammatory status of the infant. In contrast, maternal and cord plasma concentrations of n-3 FA metabolites had a mixed effect on inflammatory status in mothers and infants, which may be due to the inadequate maternal dietary intake of n-3 FAs in our study population. CONCLUSIONS: Our results reveal that maternal FA status may have an intergenerational impact on the inflammatory status of the infant. Additional research is needed to identify how dietary interventions that modify maternal FA intake prior to or during pregnancy may impact maternal and infant inflammatory status and associated long-term health outcomes.

Hypertension Related Co-Morbidities and Complications in Women of Sub-Saharan Africa: A Brief Review.

Hypertension is the leading cause of cardiovascular disease in women, and sub-Saharan African (SSA) countries have some of the highest rates of hypertension in the world. Expanding knowledge of causes, management, and awareness of hypertension and its co-morbidities worldwide is an effective strategy to mitigate its harms, decrease morbidities and mortality, and improve individual quality of life. Hypertensive disorders of pregnancy (HDPs) are a particularly important subset of hypertension, as pregnancy is a major stress test of the cardiovascular system and can be the first instance in which cardiovascular disease is clinically apparent. In SSA, women experience a higher incidence of HDP compared with other African regions. However, the region has yet to adopt treatment and preventative strategies for HDP. This delay stems from insufficient awareness, lack of clinical screening for hypertension, and lack of prevention programs. In this brief literature review, we will address the long-term consequences of hypertension and HDP in women. We evaluate the effects of uncontrolled hypertension in SSA by including research on heart disease, stroke, kidney disease, peripheral arterial disease, and HDP. Limitations exist in the number of studies from SSA; therefore, we will use data from countries across the globe, comparing and contrasting approaches in similar and dissimilar populations. Our review highlights an urgent need to prioritize public health, clinical, and bench research to discover cost-effective preventative and treatment strategies that will improve the lives of women living with hypertension in SSA.

Omega-3 Polyunsaturated Fatty Acid Levels in Maternal and Cord Plasma Are Associated with Maternal Socioeconomic Status.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) play a crucial role in fetal growth and neurodevelopment, while omega-6 (n-6) PUFAs have been associated with adverse pregnancy outcomes. Previous studies have demonstrated that socioeconomic status (SES) influences dietary intake of n-3 and n-6 PUFAs, but few studies have evaluated the association between maternal and cord plasma biomarkers of PUFAs and socioeconomic markers. An IRB-approved study enrolled mother-infant pairs (n = 55) at the time of delivery. Maternal and cord plasma PUFA concentrations were analyzed using gas chromatography. Markers of SES were obtained from validated surveys and maternal medical records. Mann-Whitney U tests and linear regression models were utilized for statistical analysis. Maternal eicosapentaenoic acid (EPA) (p = 0.02), cord EPA (p = 0.04), and total cord n-3 PUFA concentrations (p = 0.04) were significantly higher in college-educated mothers vs. mothers with less than a college education after adjustment for relevant confounders. Insurance type and household income were not significantly associated with n-3 or n-6 PUFA plasma concentrations after adjustment. Our findings suggest that mothers with lower educational status may be at risk of lower plasma concentrations of n-3 PUFAs at delivery, which could confer increased susceptibility to adverse pregnancy and birth outcomes.

Plasma Retinol Concentrations and Dietary Intakes of Mother-Infant Sets in Singleton versus Twin Pregnancy.

Vitamin A (retinol) is essential for normal fetal development, but the recommendation for maternal dietary intake (Retinol Activity Equivalent, RAE) does not differ for singleton vs. twin pregnancy, despite the limited evaluation of retinol status. Therefore, this study aimed to evaluate plasma retinol concentrations and deficiency status in mother-infant sets from singleton vs. twin pregnancies as well as maternal RAE intake. A total of 21 mother-infant sets were included (14 singleton, 7 twin). The HPLC and LC-MS/HS evaluated the plasma retinol concentration, and data were analyzed using the Mann-Whitney U test. Plasma retinol was significantly lower in twin vs. singleton pregnancies in both maternal (192.2 vs. 312.1 vs. mcg/L, p = 0.002) and umbilical cord (UC) samples (102.5 vs. 154.4 vs. mcg/L, p = 0.002). The prevalence of serum-defined vitamin A deficiency (VAD) <200.6 mcg/L was higher in twins vs. singletons for both maternal (57% vs. 7%, p = 0.031) and UC samples (100% vs. 0%, p < 0.001), despite a similar RAE intake (2178 vs. 1862 mcg/day, p = 0.603). Twin pregnancies demonstrated a higher likelihood of vitamin A deficiency in mothers, with an odds ratio of 17.3 (95% CI: 1.4 to 216.6). This study suggests twin pregnancy may be associated with VAD deficiency. Further research is needed to determine optimal maternal dietary recommendations during twin gestation.

Retinol and Pro-Vitamin A Carotenoid Nutritional Status during Pregnancy Is Associated with Newborn Hearing Screen Results.

The prenatal period is critical for auditory development; thus, prenatal influences on auditory development may significantly impact long-term hearing ability. While previous studies identified a protective effect of carotenoids on adult hearing, the impact of these nutrients on hearing outcomes in neonates is not well understood. The purpose of this study is to investigate the relationship between maternal and umbilical cord plasma retinol and carotenoid concentrations and abnormal newborn hearing screen (NHS) results. Mother-infant dyads (n = 546) were enrolled at delivery. Plasma samples were analyzed using HPLC and LC-MS/MS. NHS results were obtained from medical records. Statistical analysis utilized Mann-Whitney U tests and logistic regression models, with p ≤ 0.05 considered statistically significant. Abnormal NHS results were observed in 8.5% of infants. Higher median cord retinol (187.4 vs. 162.2 µg/L, p = 0.01), maternal trans-ß-carotene (206.1 vs. 149.4 µg/L, p = 0.02), maternal cis-ß-carotene (15.9 vs. 11.2 µg/L, p = 0.02), and cord trans-ß-carotene (15.5 vs. 8.0 µg/L, p = 0.04) were associated with abnormal NHS. Significant associations between natural log-transformed retinol and ß-carotene concentrations and abnormal NHS results remained after adjustment for smoking status, maternal age, and corrected gestational age. Further studies should investigate if congenital metabolic deficiencies, pesticide contamination of carotenoid-rich foods, maternal hypothyroidism, or other variables mediate this relationship.

Something Smells Fishy: How Lipid Mediators Impact the Maternal-Fetal Interface and Neonatal Development.

Normal pregnancy relies on inflammation for implantation, placentation, and parturition, but uncontrolled inflammation can lead to poor maternal and infant outcomes. Maternal diet is one modifiable factor that can impact inflammation. Omega-3 and -6 fatty acids obtained through the diet are metabolized into bioactive compounds that effect inflammation. Recent evidence has shown that the downstream products of omega-3 and -6 fatty acids may influence physiology during pregnancy. In this review, the current knowledge relating to omega-3 and omega-6 metabolites during pregnancy will be summarized.

Cross-sectional survey of parental barriers to participation in pediatric participant research registries.

Research registries are a powerful tool for boosting recruitment into clinical trials. However, little is known about how parents approach the decision to enroll their child in a pediatric participant research registry (PPRR). We conducted in-person, written, or telephone surveys with parents/guardians of children hospitalized at Children's Hospital of Omaha, Nebraska to identify attitudes towards and barriers to enrollment in PPRRs. Overall, our population (N = 36) had positive attitudes toward PPRRs, with 77.8% (CI: 61.6, 88.4) of participants stating they were "somewhat" or "very" likely to enroll their child. Likelihood to enroll differed between various recruitment and enrollment methods, with participants stating they would be more likely to enroll their child in a PPRR if they were recruited by their child's primary care provider or a nurse in clinic (p = 0.02) and less likely to enroll if they were recruited through social media (p<0.001). Additionally, over 90% of participants who were likely to enroll their child in a PPRR (N = 28) were also willing to provide demographic, medical, and lifestyle information. However, these participants remained concerned about inappropriate sharing of their information with insurance or for-profit companies (53.6%, CI: 35.8, 70.4) and about receiving unwanted telephone calls from the registry (78.6%, CI: 60.0, 90.0). Parents are generally willing to enroll their child in a PPRR. However, to optimize enrollment, investigators must understand parental preferences for and concerns surrounding enrollment in a PPRR.

Racial disparities in caesarean delivery among nulliparous women that delivered at term: cross-sectional decomposition analysis of Nebraska birth records from 2005-2014.

BACKGROUND: Previous studies suggest higher rates of caesarean section among women who identify as racial/ethnic minorities. The objective of this study was to understand factors contributing to differences in caesarean rates across racial and ethnic groups. METHODS: Data was collected from 2005 to 2014 Nebraska birth records on nulliparous, singleton births occurring on or after 37 weeks gestation (n = 87,908). Risk ratios (RR) and 95% confidence intervals (CI) for caesarean were calculated for different racial and ethnic categories, adjusting for maternal age, marital status, county of residence, education, insurance status, pre-pregnancy BMI, and smoking status. Fairlie decomposition technique was utilized to quantify the contribution of individual variables to the observed differences in caesarean. RESULTS: In the adjusted analysis, relative to non-Hispanic (NH) White race, both Asian-NH (RR 1.21, 95% CI 1.14, 1.28) and Black-NH races (RR 1.13, 95% CI 1.08, 1.19) were associated with a significantly higher risk for caesarean. The decomposition analysis showed that among the variables assessed, maternal age, education, and pre-pregnancy BMI contributed the most to the observed differences in caesarean rates across racial/ethnic groups. CONCLUSION: This analysis quantified the effect of social and demographic factors on racial differences in caesarean delivery, which may guide public health interventions aimed towards reducing racial disparities in caesarean rates. Interventions targeted towards modifying maternal characteristics, such as reducing pre-pregnancy BMI or increasing maternal education, may narrow the gap in caesarean rates across racial and ethnic groups. Future studies should determine the contribution of physician characteristics, hospital characteristics, and structural determinants of health towards racial disparities in caesarean rates.

Omega-6 and Omega-3 Fatty Acid-Derived Oxylipins from the Lipoxygenase Pathway in Maternal and Umbilical Cord Plasma at Delivery and Their Relationship with Infant Growth.

Omega-3 and omega-6 fatty acids are important for neonatal development and health. One mechanism by which omega-3 and omega-6 fatty acids exert their effects is through their metabolism into oxylipins and specialized pro-resolving mediators. However, the influence of oxylipins on fetal growth is not well understood. Therefore, the objective of this study was to identify oxylipins present in maternal and umbilical cord plasma and investigate their relationship with infant growth. Liquid chromatography-tandem mass spectrometry was used to quantify oxylipin levels in plasma collected at the time of delivery. Spearman's correlations highlighted significant correlations between metabolite levels and infant growth. They were then adjusted for maternal obesity (normal body mass index (BMI: ≤30 kg/m2) vs. obese BMI (>30 kg/m2) and smoking status (never vs. current/former smoker) using linear regression modeling. A p-value < 0.05 was considered statistically significant. Our study demonstrated a diverse panel of oxylipins from the lipoxygenase pathway present at the time of delivery. In addition, both omega-3 and omega-6 oxylipins demonstrated potential influences on the birth length and weight percentiles. The oxylipins present during pregnancy may influence fetal growth and development, suggesting potential metabolites to be used as biomarkers for infant outcomes.

CRATES: A one-step assembly method for Class 2 CRISPR arrays.

CRISPR-Cas systems naturally rely on CRISPR arrays to achieve immunity against multiple foreign invaders, where these arrays are also being utilized for multiplexed targeting as part of CRISPR technologies. However, CRISPR arrays have proven difficult to synthesize or assemble to-date due to the repetitive DNA repeats in each array. To overcome this barrier, we recently reported a cloning method we term CRATES (CRISPR Assembly through Trimmed Ends of Spacers) for the single-step, efficient generation of large Class 2 CRISPR arrays. CRATES generates CRISPR arrays through assembly of multiple repeat-spacer subunits using defined junction sequences within the trimmed portion of the CRISPR spacers. These arrays can be utilized by single-effector nucleases associated with Class 2 CRISPR-Cas systems, such as Cas9, Cas12a/Cpf1, or Cas13a/C2c2. Here, we describe in detail the steps for generating arrays utilized by Cas9 and Cas12a as well as composite arrays co-utilized by both nucleases. We also generate a representative three-spacer array and demonstrate multiplexed DNA cleavage through plasmid-clearance assays in Escherichia coli. This method is expected to simplify the study of natural CRISPR arrays and facilitate multiplexed targeting with programmable nucleases from Class 2 Cas nucleases across the myriad applications of CRISPR technologies.

Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.

The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.

Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing.

Filamentous fungi produce varieties of natural products even in a strain dependent manner. However, the genetic basis of chemical speciation between strains is still widely unknown. One example is trypacidin, a natural product of the opportunistic human pathogen Aspergillus fumigatus, which is not produced among different isolates. Combining computational analysis with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool for advanced molecular genetic studies in filamentous fungi, exploiting selectable markers separated from the edited locus.

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.