RNA-based logic for selective protein expression in senescent cells.

Cellular senescence is a cellular state characterized by irreversible growth arrest, resistance to apoptosis and secretion of inflammatory molecules, which is causally linked to the pathogenesis of many age-related diseases. Besides, there is accumulating evidence that selective removal of senescent cells can benefit therapies for cancer and fibrosis by modulating the inflammatory microenvironment. While the field of so-called senolytics has spawned promising small molecules and peptides for the selective removal of senescent cells, there is still no effective means to detect senescent cells in vivo, a prerequisite for understanding the role of senescence in pathophysiology and to assess the effectiveness of treatments aimed at removing senescent cells. Here, we present a strategy based on an mRNA logic circuit, that yields mRNA-dependent protein expression only when a senescence-specific miRNA signature is present. Following a validation of radiation-induced senescence induction in primary human fibroblasts, we identify miRNAs up- and downregulated in association with cellular senescence using RT-qPCR. Incorporating binding sites to these miRNAs into the 3' untranslated regions of the mRNA logic circuit, we demonstrate the senescence-specific expression of EGFP for detection of senescent cells and of a constitutively active caspase-3 for selective removal. Altogether, our results pave the way for a novel approach to execute an mRNA-based programme specifically in senescent cells aimed at their detection or selective removal.

Unraveling mRNA delivery bottlenecks of ineffective delivery vectors by co-transfection with effective carriers.

The messenger RNA (mRNA) SARS-CoV-2 vaccines have demonstrated the therapeutic potential of this novel drug modality. Protein expression is the consequence of a multistep delivery process that relies on proper packaging into nanoparticle carriers to protect the mRNA against degradation enabling effective cellular uptake and endosomal release, and liberating the mRNA in the cytosol. Bottlenecks along this route remain challenging to pinpoint. Although methods to assess endosomal escape of carriers have been developed, versatile strategies to identify bottlenecks along the delivery trajectory are missing. Here, it is shown that co-incubating an inefficient nanoparticle formulation with an efficient one solves this problem. Cells were co-incubated with mRNA nanoparticles formed with either the efficient cell-penetrating peptide (CPP) PepFect14 or the inefficient CPP nona-arginine (R9). Co-transfection enhanced cellular uptake and endosomal escape of R9-formulated mRNA, resulting in protein expression, demonstrating that both vectors enter cells along the same route. In addition, cells were transfected with a galectin-9-mCherry fusion protein to detect endosomal rupture. Remarkably, despite endosomal release, mRNA remained confined to punctate structures, identifying mRNA liberation as a further bottleneck. In summary, co-transfection offers a rapid means to identify bottlenecks in cytosolic mRNA delivery, supporting the rational design and optimization of intracellular mRNA delivery systems.

Impact of conditional cash transfer programmes on antenatal care service uptake in low and middle-income countries: a systematic review.

OBJECTIVE: Antenatal care (ANC) is crucial to protecting the health of pregnant women and their unborn children; however, the uptake of ANC among pregnant women in low and middle-income countries (LMICs) is suboptimal. One popular strategy to increase the uptake of health services, including ANC visits, are conditional cash transfer (CCT) programmes. CCT programmes require beneficiaries to comply with certain conditionalities in order to receive a financial sum. A systematic review was carried out to determine whether CCT programmes have a positive impact on ANC uptake in LMIC populations. METHODS: Electronic databases CENTRAL, MEDLINE, Embase, Maternity and Infant Care and Global Health were searched from database inception to 21 January 2022. Reference checking and grey literature searches were also applied. Eligible study designs were randomised controlled trials, controlled before-after studies and interrupted time series analysis. Risk of bias assessments were undertaken for each study by applying the Risk of Bias 2 tool and the Risk of Bias in Non-randomised Studies of Interventions tool. RESULTS: Out of 1534 screened articles, 18 publications were included for analysis. Eight studies reported statistically non-significant results on all reported outcomes. Seven studies demonstrated statistically significant positive effects ranging from 5.5% to 45% increase in ANC service uptake. A further three studies reported small but statistically significant impact of CCT on the use of ANC services in both positive (2.5% increase) and negative (3.7% decrease) directions. Subanalysis of results disaggregated by socioeconomic status (SES) indicated that ANC attendance may be more markedly improved by CCT programmes in low SES populations; however, results were inconclusive. CONCLUSION: Our evidence synthesis presented here demonstrated a highly heterogeneous evidence base pertaining to the impact of CCTs on ANC attendance. More high-powered studies are required to elucidate the true impact of CCT programmes on ANC uptake, with particular focus on the barriers and enablers of such programmes in achieving intended outcomes.