Central and peripheral delivered AAV9-SMN are both efficient but target different pathomechanisms in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene and low SMN protein levels. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy and clinical trials use a single intravenous bolus of the blood-brain-barrier penetrant scAAV9-cba-SMN by either systemic or central nervous system (CNS) delivery, resulting in impressive amelioration of the clinical phenotype but not a complete cure. The impact of scAAV9-cba-SMN treatment regimens on the CNS as well as on specific peripheral organs is yet to be described in a comparative manner. Therefore, we injected SMA mice with scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. In our system, ICV injections increased SMN in peripheral organs and the CNS while IV administration increased SMN in peripheral tissues only, largely omitting the CNS. Both treatments rescued several peripheral phenotypes while only ICV injections were neuroprotective. Surprisingly, both delivery routes resulted in a robust rescue effect on survival, weight, and motor function, which in IV-treated mice relied on peripheral SMN restoration but not on targeting the motor neurons. This demonstrates the independent contribution of peripheral organs to SMA pathology and suggests that treatments should not be restricted to motor neurons.

Digital immunoassay for biomarker concentration quantification using solid-state nanopores.

Single-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores-fully electronic sensors with single-molecule sensitivity-are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence ("1") or absence ("0") of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.

Characterization of immune cells and cytokine localization in the rat utero-placental unit mid- to late gestation.

The success of pregnancy is dependent on the precise regulation of the immune response within the utero-placental environment. Rats are beginning to be widely used as a model for human immune-related pregnancy complications. However, our knowledge of immune cells and cytokine localization in the rat utero-placental tissue is limited. The current study aimed to localize the immune cell populations, including uterine natural killer (uNK) cells, neutrophils, and macrophages within the rat utero-placental unit at two crucial gestational ages, gestational days 15.5 and 18.5. In addition, we characterized the distribution of the cytokines TNFα, IFNγ, and IL-10 in the utero-placental regions at both the above-mentioned gestational ages. Our study has demonstrated co-localization TNFα and IFNγ with uNK cells in perivascular regions of the rat mesometrial triangle at both gestational ages. Neutrophils and IL-10-positive cells were localized at the maternal-fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15.5 to GD18.5, which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The current study furthers our knowledge of the localization of uterine immune cells and relevant cytokines, and provides a base from which to research the function of these immune cells and cytokines during rat pregnancy as a model to study human immune-related pregnancy complications.

Uterine Spiral Artery Remodeling: The Role of Uterine Natural Killer Cells and Extravillous Trophoblasts in Normal and High-Risk Human Pregnancies.

The process of uterine spiral artery remodeling in the first trimester of human pregnancy is an essential part of establishing adequate blood perfusion of the placenta that will allow optimal nutrient/waste exchange to meet fetal demands during later development. Key regulators of spiral artery remodeling are the uterine natural killer cells and the invasive extravillous trophoblasts. The functions of these cells as well as regulation of their activation states and temporal regulation of their localization within the uterine tissue are beginning to be known. In this review, we discuss the roles of these two cell lineages in arterial remodeling events, their interaction/influence on one another and the outcomes of altered temporal, and spatial regulation of these cells in pregnancy complications.

Essential role for the SLK protein kinase in embryogenesis and placental tissue development.

BACKGROUND: Over the past decade, the Ste20-like kinase SLK, has been implicated in several signaling processes. SLK repression has been shown to impair cell cycle kinetics and inhibit FAK-mediated cell migration. Here, using a gene trapped allele, we have generated mice expressing a truncated form of the SLK kinase. RESULTS: Our results show that an SLK-LacZ fusion protein is expressed in embryonic stem cells and in embryos throughout development. We find that the SLK-LacZ fusion protein is less efficient at phosphorylating substrates resulting in reduced cell proliferation within the embryos and angiogenic defects in the placentae of the homozygous mutant animals at embryonic day (E) 12.5. This results in marked developmental defects and apoptotic lesions in the embryos by E14.5. CONCLUSIONS: Homozygotes expressing the SLK-LacZ fusion protein present with an embryonic lethal phenotype occurring between E12.5 and E14.5. Overall, we demonstrate a requirement for SLK kinase activity in the developing embryo and placenta.

Trophoblast invasion and blood vessel remodeling are altered in a rat model of lifelong maternal obesity.

Maternal obesity is associated with an increased risk of a number of pregnancy complications, including fetal demise, which may be linked to impaired placental development as a result of altered trophoblast invasion and vessel remodeling. Therefore, we examined these parameters in pregnant rats fed a control (normal weight) or high fat (HF) diet (obese) at 2 critical times of rat placental development. Early trophoblast invasion was increased by approximately 2-fold in HF-fed dams with a concomitant increase in the expression of matrix metalloproteinase 9 protein, a mediator of tissue remodeling and invasion. Furthermore, we observed significantly higher levels of smooth muscle actin surrounding the placental spiral arteries of HF-fed dams, suggesting impaired spiral artery remodeling. Taken together, the results of this study suggest that altered placental development is an important contributor to the poor pregnancy outcomes and increased fetal demise in our model of lifelong maternal obesity.