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.

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.

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.