Lipid nanoparticle structure and delivery route during pregnancy dictate mRNA potency, immunogenicity, and maternal and fetal outcomes.

Treating pregnancy-related disorders is exceptionally challenging because the threat of maternal and/or fetal toxicity discourages the use of existing medications and hinders new drug development. One potential solution is the use of lipid nanoparticle (LNP) RNA therapies, given their proven efficacy, tolerability, and lack of fetal accumulation. Here, we describe LNPs for efficacious mRNA delivery to maternal organs in pregnant mice via several routes of administration. In the placenta, our lead LNP transfected trophoblasts, endothelial cells, and immune cells, with efficacy being structurally dependent on the ionizable lipid polyamine headgroup. Next, we show that LNP-induced maternal inflammatory responses affect mRNA expression in the maternal compartment and hinder neonatal development. Specifically, pro-inflammatory LNP structures and routes of administration curtailed efficacy in maternal lymphoid organs in an IL-1ß-dependent manner. Further, immunogenic LNPs provoked the infiltration of adaptive immune cells into the placenta and restricted pup growth after birth. Together, our results provide mechanism-based structural guidance on the design of potent LNPs for safe use during pregnancy.

Advances in proteomics methods for the analysis of exhaled breath condensate.

The analysis of exhaled breath condensate (EBC) demonstrates a promising avenue of minimally invasive biopsies for diagnostics. EBC is obtained by cooling exhaled air and collecting the condensation to be utilized for downstream analysis using various analytical methods. The aqueous phase of breath contains a large variety of miscible small compounds including polar electrolytes, amino acids, cytokines, chemokines, peptides, small proteins, metabolites, nucleic acids, and lipids/eicosanoids-however, these analytes are typically present at minuscule levels in EBC, posing a considerable technical challenge. Along with recent improvements in devices for breath collection, the sensitivity and resolution of liquid chromatography coupled to online mass spectrometry-based proteomics has attained subfemtomole sensitivity, vastly enhancing the quality of EBC sample analysis. As a result, proteomics analysis of EBC has been expanding the field of breath biomarker research. We present an au courant overview of the achievements in proteomics of EBC, the advancement of EBC collection devices, and the current and future applications for EBC biomarker analysis.

Gene body DNA hydroxymethylation restricts the magnitude of transcriptional changes during aging.

DNA hydroxymethylation (5hmC), the most abundant oxidative derivative of DNA methylation, is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in age-related diseases, its functional role in aging remains unknown. Here, using mouse liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and restricts the magnitude of gene expression changes with age. Mechanistically, 5hmC decreases the binding of splicing associated factors and correlates with age-related alternative splicing events. We found that various age-related contexts, such as prolonged quiescence and senescence, drive the accumulation of 5hmC with age. We provide evidence that this age-related transcriptionally restrictive function is conserved in mouse and human tissues. Our findings reveal that 5hmC regulates tissue-specific function and may play a role in longevity.

Placental drug transport and fetal exposure during pregnancy is determined by drug molecular size, chemistry, and conformation.

Pregnant people are unable to take many prescription and over-the-counter medications because of suspected or known risk to the fetus. This undermedication contributes to the high maternal mortality rate in the United States and detracts from the quality of life of pregnant people. As such, there is an urgent need to develop safe pharmaceutical formulations for use during pregnancy. Most drugs are small molecules that easily cross the placenta, which is the biological barrier that separates the maternal and fetal bloodstreams. One potential approach to preventing fetal drug accumulation is to design drug compounds that are excluded by the placenta; however, there is little understanding of how macromolecular drug properties affect transplacental transport. To address this knowledge gap, we examined the transport behavior of fluorescently-labeled polymers with varying size, conformation, and chemistry. We compared these polymers to unconjugated fluorescein, a small molecule model drug that readily crosses biological barriers. We found that molecular size affected transplacental transport in an in vitro model, BeWo b30 monolayers, as well as in pregnant mice, with larger polymers having lower permeability. In addition to size, polymer chemistry altered behavior, with polyethylene glycol (PEG) molecules permeating the placental barrier to a greater extent than dextrans of equivalent molecular weight. PEG molecules were also more readily taken up into placental cells in vivo. These findings will inform the future development of drug conjugates or other macromolecular medicines that can safely be used during pregnancy.

Gene body DNA hydroxymethylation restricts the magnitude of transcriptional changes during aging.

DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the modification in aging remains largely unknown. Here, we report that 5hmC is stably enriched in multiple aged organs. Using the liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and thereby restricts the magnitude of gene expression changes during aging. Mechanistically, we found that 5hmC decreases binding affinity of splicing factors compared to unmodified cytosine and 5mC, and is correlated with age-related alternative splicing events, suggesting RNA splicing as a potential mediator of 5hmC's transcriptionally restrictive function. Furthermore, we show that various age-related contexts, such as prolonged quiescence and senescence, are partially responsible for driving the accumulation of 5hmC with age. We provide evidence that this age-related function is conserved in mouse and human tissues, and further show that the modification is altered by regimens known to modulate lifespan. Our findings reveal that 5hmC is a regulator of tissue-specific function and may play a role in regulating longevity.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Systemic Immunosuppression and Risk of Endophthalmitis After Intravitreal Anti-Vascular Endothelial Growth Factor Injections.

BACKGROUND AND OBJECTIVE: To evaluate the impact of systemic immunosuppressive therapy on the rates and outcomes of endophthalmitis following intravitreal anti-vascular endothelial growth factor (VEGF) injections. PATIENTS AND METHODS: A retrospective, single-center, comparative cohort study examining eyes undergoing intravitreal anti-VEGF injections from January 2016 to September 2019. Cohorts were created based on concurrent immunosuppressive therapy at time of injection. RESULTS: Of 270,347 anti-VEGF injections administered, 1,300 injections (0.48%) were administered while on systemic immunosuppressive therapy. The odds of endophthalmitis occurring in the immunosuppression group was 9.86 (95% confidence interval [CI], 4.0-24.3; P < .001) times higher than the no-immunosuppression group. Symptom onset occurred 2.51 (95% CI, 0.15-4.870; P = .040) days earlier in the immunosuppression cohort; visual acuity at 6 months after treatment was similar in both groups. CONCLUSIONS: Patients on systemic immunosuppressive medications undergoing intravitreal injections may be at increased risk for post-injection endophthalmitis and may have earlier symptom onset. However, visual outcomes were similar between the two groups. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:S17-S22.].