The timing of antenatal care access for adolescent pregnancies in Cape Town, South Africa.

Background:  Late antenatal care (ANC)-seeking among pregnant adolescents threatens their health outcomes, and the health outcomes of their new-borns. South Africa has experienced a rapid increase in adolescent pregnancies during the COVID-19 pandemic, adding to the existing concerns around adolescent pregnancy care-seeking behaviour.

Aim:  The main aim of this study was to investigate the causes and covariates of late ANC access among adolescents in the Cape Town Metropole, South Africa.

A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs.

While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3.

'The nurse did not even greet me': how informed versus non-informed patients evaluate health systems responsiveness in South Africa.

Introduction: Universal Health Coverage is not only about access to health services but also about access to high-quality care, since poor experiences may deter patients from accessing care. Evidence shows that quality of care drives health outcomes, yet little is known about non-clinical dimensions of care, and patients' experience thereof relative to satisfaction with visits. This paper investigates the role of non-clinical dimensions of care in patient satisfaction.

CRISPR-mediated transcriptional activation with synthetic guide RNA.

The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa.

Use of simulated patients to assess hypertension case management at public healthcare facilities in South Africa.

Objective: Our study aims to evaluate hypertensive case management in South Africa's public health sector using simulated patients.

Method: Our study describes interactions between hypertensive simulated patients and primary healthcare workers at 39 public sector healthcare facilities in two metropolitan centres in the Eastern and Western Cape Provinces of South Africa. Our analysis focus on 97 interactions where our eight simulated patients tested within range for stage 1 hypertension, that is with SBP 140-159 mmHg and/or DBP 90-99 mmHg.

Demand-Side Causes and Covariates of Late Antenatal Care Access in Cape Town, South Africa.

Objectives The objective of this study was to investigate the causes and covariates of late antenatal care access in South Africa. Methods A cross-sectional study was conducted, interviewing 221 women at four public-sector labour wards in Cape Town, South Africa in 2014. A definition of late attendance as attending ≥ 5 months was used.

The rise and fall of mortality inequality in South Africa in the HIV era.

Post-apartheid South Africa has seen an unprecedented rise and fall of mortality in less than two decades as a result of the HIV/AIDS epidemic and the subsequent rollout of free antiretroviral therapy (ART). Since the incidence of both was not equal for rich and poor, it is likely to also have affected disparities in health and survival chances by income. We use large nationwide surveys for 2001, 2007 and 2011 to obtain estimates of average income and mortality at the aggregate level of a municipality, and then to examine changes in mortality - and in inequality in mortality by income ─ over time.

Measuring Quality Gaps in TB Screening in South Africa Using Standardised Patient Analysis.

This is the first multi-district Standardised Patient (SP) study in South Africa. It measures the quality of TB screening at primary healthcare (PHC) facilities. We hypothesise that TB screening protocols and best practices are poorly adhered to at the PHC level.

Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L.

A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2.

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications.

A comparable yardstick: adjusting for education bias in South African health system responsiveness ratings.

Health-system responsiveness (HSR) measures the experience of health-system users in terms of the non-clinical aspects of the health system. This has been operationalized as a measurable construct in multiple surveys and studies. According to the literature, reporting behaviour may vary systematically across socio-demographic characteristics.

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs.

High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle.

Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments.

Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments.

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.

The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology.