Chemical modification patterns for microRNA therapeutic mimics: a structure-activity relationship (SAR) case-study on miR-200c.

microRNA (miRNA) mimics are an emerging class of oligonucleotide therapeutics, with a few compounds already in clinical stages. Synthetic miRNAs are able to restore downregulated levels of intrinsic miRNAs, allowing for parallel regulation of multiple genes involved in a particular disease. In this work, we examined the influence of chemical modifications patterns in miR-200c mimics, assessing the regulation of a selection of target messenger RNAs (mRNA) and, subsequently, of the whole transcriptome in A549 cells. We have probed 37 mimics and provided an initial set of instructions for designing miRNA mimics with potency and selectivity similar to an unmodified miRNA duplex. Additionally, we have examined the stability of selected mimics in serum. Finally, the selected two modification patterns were translated to two other miRNAs, miR-34a and miR-155. To differing degrees, these designs acted on target mRNAs in a similar manner to the unmodified mimic. Here, for the first time, we describe a structured overview of 'miRNA mimics modification templates' that are chemically stabilised and optimised for use in an in vitro set up and highlight the need of further sequence specific optimization when mimics are to be used beyond in vitro tool experiments.

Doppler Echocardiographic Phenotypes in Suspected 'Severe' Aortic Stenosis: Matrix-Based Approach to Diagnosis and Management.

BACKGROUND: Among patients with suspected severe aortic stenosis (AS), Doppler echocardiographic (DE) data are often discordant, and further analysis is required for accurate diagnosis and optimal management. In this study, an automated matrix-based approach was applied to an echocardiographic database of patients with AS that identified 5 discrete echocardiographic data patterns, 1 concordant and 4 discordant, each reflecting a particular pathophysiology/measurement error that guides further workup and management. METHODS: A primary/discovery cohort of consecutive echocardiographic studies with at least 1 DE parameter of severe AS and analogous data from an independent secondary/validation cohort were retrospectively analyzed. Parameter thresholds for inclusion were aortic valve area (AVA) <1.0 cm2, transaortic mean gradient (MG) ≥ 40 mmHg, and/or transaortic peak velocity (PV) ≥ 4.0 m/sec. Doppler velocity index (DVI) was also determined. Logic provided by an in-line SQL query embedded within the database was used to assign each patient to 1 of 5 discrete matrix patterns, each reflecting 1 or more specific pathophysiologies. Feasibility of automated pattern-driven triage of discordant cases was also evaluated. RESULTS: In both cohorts, data from each patient fitted only 1 data pattern. Of the 4,643 primary cohort patients, 39% had concordant parameters for severe AS and DVI <0.30 (pattern 1); 35% had AVA < 1.0 cm2, MG < 40 mm Hg, PV < 4 m/sec, DVI < 0.30 (pattern 2); 9% had MG ≥ 40 mmHg and/or PV ≥ 4 m/sec, DVI > 0.30 (pattern 3); 10% had AVA < 1.0 cm2, MG < 40 mmHg, PV < 4 m/sec, DVI >0.30 (pattern 4); and 7% had MG > 40 mmHg and/or PV ≥ 4 m/sec, AVA > 1.0 cm2, DVI < 0.30 (pattern 5). Findings were validated among the 387 secondary cohort patients in whom pattern distribution was remarkably similar. CONCLUSIONS: Matrix-based pattern recognition permits automated in-line identification of specific pathophysiology and/or measurement error among patients with suspected severe AS and discordant DE data.

Evaluation of commercial devices for patient specific QA of stereotactic radiotherapy plans.

Stereotactic radiotherapy (SRT) methods have become common for the treatment of small tumors in various parts of the body. Small field dosimetry has a unique set of challenges when it comes to the pre-treatment validation of a radiotherapy plan that involves film dosimetry or high-resolution detectors. Comparison of commercial quality assurance (QA) devices to the film dosimetry method for pre-treatment evaluation of stereotactic radiosurgery (SRS), fractionated SRT, and stereotactic body radiation therapy treatment plans have been evaluated in this study. Forty stereotactic QA plans were measured using EBT-XD film, IBA Matrixx Resolution, SNC ArcCHECK, Varian aS1200 EPID, SNC SRS MapCHECK, and IBA myQA SRS. The results of the commercial devices are compared to the EBT-XD film dosimetry results for each gamma criteria. Treatment plan characteristics such as modulation factor and target volume were investigated for correlation with the passing rates. It was found that all detectors have greater than 95% passing rates at 3%/3 mm. Passing rates decrease rapidly for ArcCHECK and the Matrixx as criteria became more strict. In contrast, EBT-XD film, SNC SRS MapCHECK, and IBA myQA SRS passing rates do not decline as rapidly when compared to Matrix Resolution, ArcCHECK, and the EPID. EBT-XD film, SNC SRS MapCHECK, and IBA myQA SRS maintain greater than 90% passing rate at 2%/1 mm and greater than 80% at 1%/1 mm. Additionally, the ability of these devices to detect changes in dose distribution due to MLC positioning errors was investigated. Ten VMAT SBRT/SRS treatment plans were created with 6 MV FFF or 10 MV FFF beam energies using Eclipse 15.6. A MATLAB script was used to create two MLC positioning error scenarios from the original treatment plan. It was found that errors in MLC positioning were most reliably detected at 2%/1 mm for high-resolution detectors and that lower-resolution detectors did not consistently detect MLC positioning errors.

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR.

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.

Reassessment of weak parent-of-origin expression bias shows it rarely exists outside of known imprinted regions.

In mouse and human, genes subjected to genomic imprinting have been shown to function in development, behavior, and post-natal adaptations. Failure to correctly imprint genes in human is associated with developmental syndromes, adaptive, and metabolic disorders during life as well as numerous forms of cancer. In recent years researchers have turned to RNA-seq technologies applied to reciprocal hybrid strains of mice to identify novel imprinted genes, causing a threefold increase in genes reported as having a parental origin-specific expression bias. The functional relevance of parental origin-specific expression bias is not fully appreciated especially since many are reported with only minimal parental bias (e.g. 51:49). Here, we present an in-depth meta-analysis of previously generated RNA-seq data and show that the methods used to generate and analyze libraries greatly influence the calling of allele-specific expression. Validation experiments show that most novel genes called with parental-origin-specific allelic bias are artefactual, with the mouse strain contributing a larger effect on expression biases than parental origin. Of the weak novel genes that do validate, most are located at the periphery of known imprinted domains, suggesting they may be affected by local allele- and tissue-specific conformation. Together these findings highlight the need for robust tools, definitions, and validation of putative imprinted genes to provide meaningful information within imprinting databases and to understand the functional and mechanistic implications of the process.

Comparative Outcomes of TAVR in Mixed Aortic Valve Disease and Aortic Stenosis: A Meta-analysis.

INTRODUCTION: Transcatheter aortic valve replacement (TAVR) has become a suitable alternative to surgical aortic valve replacement (SAVR) for the treatment of symptomatic severe aortic stenosis (AS). A high proportion of patients with AS have mixed aortic valve disease (MAVD) with mild or more concurrent aortic regurgitation (AR). Differential outcomes of TAVR among patients with AS and MAVD have not been well characterized. We compared 1-year mortalities following TAVR among patients with MAVD and AS. METHODS: We conducted a meta-analysis of studies published in PubMed/Medline. The primary outcome was 1-year all-cause mortality following TAVR among patients with MAVD vs. AS. Secondary endpoints were: (1) incidence of AR within 30 days following TAVR (post TAVR AR); and (2) 1-year all-cause mortality within each group stratified according to severity of post TAVR AR. RESULTS: Nine studies involving 9505 participants were included in the analysis. At 1 year following TAVR, mortality was lower in MAVD than in AS; HR 0.89, 95% CI 0.81-0.98. The mortality advantage increased when pre-TAVR AR was moderate or more; HR 0.84, 95% CI 0.72-0.99. The mortality advantage was attenuated after correction for publication bias. There was a higher risk of post TAVR AR in the MAVD group; OR 1.51, 95% CI 1.20-1.90 but the impact on mortality of moderate vs. mild post TAVR AR was greater among patients with AS than in patients with MAVD HR 1.67 95% CI 0.89-3.14 vs. 0.93 95% CI 0.47-1.85. CONCLUSIONS: Patients with MAVD have similar or improved survival 1 year after TAVR compared to those with AS.

A rapidly enlarging giant pericardial cyst resected by video-assisted thoracoscopic surgery (VATS): A case report.

Pericardial cysts are rare mediastinal masses of congenital etiology. Giant pericardial cysts measuring greater than 10 cm are even rarer. In a small proportion of cases, the natural history of pericardial cyst is one of continuous slow growth. Symptomatic pericardial cysts can be treated initially with percutaneous aspiration. Very large or complicated cysts are preferentially treated by open surgical excision. We present a case of a rapidly growing giant pericardial cyst in a 36-year-old male. The cyst was an overlooked incidental finding on a computed tomography scan of the abdomen and pelvis obtained for unrelated reasons seven years prior. At that time, it measured 4 × 2 × 1 cm. No further evaluation was carried out until he became symptomatic, at which time the cyst had a more than 2-fold increase in maximum diameter to a size of 11 × 10 × 6 cm. This resulted in compression of adjacent cardiac structures without hemodynamic sequalae. The cyst was completely excised via video-assisted thoracoscopic surgery, facilitated by initial intra-operative needle aspiration to reduce the size for safe mobilization. .

Brachytherapy for Central Serous Chorioretinopathy.

Brachytherapy is widely used for the treatment of choroidal melanoma and has recently been explored for the treatment of wet age-related macular degeneration. We propose the use of low dose radiation via episcleral brachytherapy in refractory cases of central serous chorioretinopathy (CSCR). The pathogenesis of CSCR involves dilatation and hyperpermeability of large choroidal vessels. Low dose radiation can induce intimal proliferation in large choroidal vessels and decrease their hyperpermeability. Concerns about the use of brachytherapy in CSCR include damage to the choriocapillaris or the retinal vessels. This can be addressed with the use of a specialized device through which a very precise and appropriate dose can be delivered. The dose of the radiation delivered decreases exponentially at a depth of approximately 0.5-1.5 mm from the devise-sclera interface. Considering an increased choroidal thickness in cases of CSCR, delivery of a safe dose can be assured.

Two-year outcomes of episcleral brachytherapy adjunct to anti-VEGF therapy for treatment-resistant nAMD.

PURPOSE: This study was designed to demonstrate the safety and feasibility of episcleral brachytherapy (ESB) for the treatment of anti-vascular endothelial growth factor (anti-VEGF) resistant neovascular age-related macular degeneration (nAMD) in a 6-subject cohort adjunct to anti-VEGF therapy. METHODS: Six eyes of six subjects with anti-VEGF resistant nAMD (persistent fluid or hemorrhage despite frequent anti-VEGF treatment) were treated with ESB between May 2018 and July 2018 as part of a larger early feasibility trial. Baseline and follow-up exams with multi-modal imaging were conducted. RESULTS: In this analysis, six eyes were included. The mean age was 74.7 years; 33% were female; 67% had polypoidal choroidal vasculopathy. The mean number of lifetime anti-VEGF injections received prior to the study enrollment was 33.9 injections and 10 injections in the year prior to the study enrollment. In the first and second years following ESB, the mean number of injections was 8.5 and 8, respectively. No evidence of radiation-induced toxicity through 2 years following ESB was observed. The mean baseline VA was 55.3 letters. At 1 year, the mean VA increased by 3.2 letters and 1.7 letters at year 2. At 2 years, the mean change in vascular complex on ICGA was - 18%, - 43% on OCTA, and - 5% on FA. The subjects also experienced a mean decrease in CRT on OCT of 21% after 2 years. CONCLUSIONS: The results from this six-subject cohort with 2-year data support additional investigations of ESB for nAMD, specifically those with persistent disease activity and treatment resistant nAMD.

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

Coronary Artery Fistulae in Adult: Two Decades of Experience in Clinical Presentation, Angiographic Feature, and Management.

Coronary artery fistula (CAF) in adults is a rare but significant coronary artery anomaly. Main data on that rare disease were mostly obtained from case reports and small studies. In presented study, we share our two-decade experience on the clinical and angiographic characteristics of CAF. The data were collected retrospectively by analyzing the angiographic data between January 1, 2000 and December 31, 2019. Demographic data, clinical data, laboratory, and cardiac catheterization reports were reviewed. CAFs were found in 40 patients (0.06%). There were 22 male (55%) patients. The mean age was 61.2 years. Twenty-nine patients (72.5%) had small, 4 patients (10%) had medium, and 7 patients (17.5%) had large CAFs. The majority of study population had solitary CAF ( n = 31, 77.5%). The pulmonary artery is the major side of fistula drainage ( n = 20, 50%). The study population was divided into two groups as follow: group 1-small CAFs 29 (72.5%), group 2-medium and large CAF (MLCAF) 11 (27.5%). Patients with MLCAFs had more atrial fibrillation, abnormal coronary morphology, and multiple fistulae. In patients with hemodynamically significant CAFs, 7 (17.5%) patients had surgical ligation and 3 (7.5%) patients had transcutaneous closure. Three patients died during mean follow-up period of 5 years. The incidence and the pattern of CAFs in our study were similar to previous studies. Clinical course of small fistulae was benign. Symptomatic MLCAFs need to be treated by transcatheter or surgical way and should be individualized per patient.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment.

Placental secretome characterization identifies candidates for pregnancy complications.

Alterations in maternal physiological adaptation during pregnancy lead to complications, including abnormal birthweight and gestational diabetes. Maternal adaptations are driven by placental hormones, although the full identity of these is lacking. This study unbiasedly characterized the secretory output of mouse placental endocrine cells and examined whether these data could identify placental hormones important for determining pregnancy outcome in humans. Secretome and cell peptidome analyses were performed on cultured primary trophoblast and fluorescence-activated sorted endocrine trophoblasts from mice and a placental secretome map was generated. Proteins secreted from the placenta were detectable in the circulation of mice and showed a higher relative abundance in pregnancy. Bioinformatic analyses showed that placental secretome proteins are involved in metabolic, immune and growth modulation, are largely expressed by human placenta and several are dysregulated in pregnancy complications. Moreover, proof-of-concept studies found that secreted placental proteins (sFLT1/MIF and ANGPT2/MIF ratios) were increased in women prior to diagnosis of gestational diabetes. Thus, placental secretome analysis could lead to the identification of new placental biomarkers of pregnancy complications.

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker.

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

Coronary artery aneurysm presenting as acute coronary syndrome: two case reports and a review of the literature.

Coronary artery aneurysm (CAA) is an uncommon coronary disease, with a reported incidence in adults ranging from 0.33 to 4.9%.It is usually considered a variant of coronary artery disease (CAD). CAA is associated with thrombus formation due to abnormal laminar flow, as well as abnormal platelet and endothelial-derived pathophysiologic factors within the CAA. CAA identified in the context of acute coronary syndrome (ACS) poses several unique management challenges. Optimal antiplatelet and anticoagulant therapy is the mainstay of therapy. Percutaneous intervention for CAA is associated with complications including distal embolization of thrombus, no-reflow phenomenon, stent malposition, dissection, and rupture. There are currently no accepted guidelines to direct the management of CAA in patients presenting with ACS. Preference for conservative vs. surgical or catheter-based management is controversial. We review the literature and report different treatment strategies for two cases with both CAA and ACS.

Continuous human uterine NK cell differentiation in response to endometrial regeneration and pregnancy.

Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy.

Eosinophilic Myocarditis: Is Endomyocardial Biopsy Essential for Diagnosis in the Era of Advanced Cardiac Imaging?

A 60-year-old female presented with dyspnea and chest pressure. Clinical presentation, laboratory data, echocardiography, and cardiac magnetic resonance (CMR) imaging findings confirmed diagnosis of eosinophilic myocarditis and obviated unnecessary invasive endomyocardial biopsy. She was treated with oral steroid and oral anticoagulation. Follow-up CMR imaging showed resolution of the left ventricle thrombus with improvement in endomyocardial inflammation.

Phenotypic and functional characterization of first-trimester human placental macrophages, Hofbauer cells.

Hofbauer cells (HBCs) are a population of macrophages found in high abundance within the stroma of the first-trimester human placenta. HBCs are the only fetal immune cell population within the stroma of healthy placenta. However, the functional properties of these cells are poorly described. Aligning with their predicted origin via primitive hematopoiesis, we find that HBCs are transcriptionally similar to yolk sac macrophages. Phenotypically, HBCs can be identified as HLA-DR-FOLR2+ macrophages. We identify a number of factors that HBCs secrete (including OPN and MMP-9) that could affect placental angiogenesis and remodeling. We determine that HBCs have the capacity to play a defensive role, where they are responsive to Toll-like receptor stimulation and are microbicidal. Finally, we also identify a population of placenta-associated maternal macrophages (PAMM1a) that adhere to the placental surface and express factors, such as fibronectin, that may aid in repair.

Pilot Study: Texture Analysis of PET Imaging Demonstrates Changes in 18F-FDG Uptake of the Brain After Prophylactic Cranial Irradiation.

Prophylactic cranial irradiation (PCI) is used to decrease the probability of developing brain metastases in patients with small cell lung cancer and has been linked to deleterious cognitive effects. Although no well-established imaging markers for these effects exist, previous studies have shown that structural and metabolic changes in the brain can be detected with MRI and PET. This study used an image processing technique called texture analysis to explore whether global changes in brain glucose metabolism could be characterized in PET images. Methods: 18F-FDG PET images of the brain from patients with small cell lung cancer, obtained before and after the administration of PCI, were processed using texture analysis. Texture features were compared between the pre- and post-PCI images. Results: Multiple texture features demonstrated statistically significant differences before and after PCI when texture analysis was applied to the brain parenchyma as a whole. Regional differences were also seen but were not statistically significant. Conclusion: Global changes in brain glucose metabolism occur after PCI and are detectable using advanced image processing techniques. These changes may reflect radiation-induced damage and thus may provide a novel method for studying radiation-induced cognitive impairment.