Left atrial appendage aneurysm enlarged in the neonatal period.

We describe a newborn with a congenital left atrial appendage aneurysm. The aneurysm size did not change prenatally. However, it rapidly enlarged after birth. MRI was useful for assessing the aneurysm extent and exact size, and for diagnosis. Respiratory distress and feeding difficulties appeared, and surgery was performed. These symptoms disappeared post-operatively. The patient is alive without complications or recurrence.

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic-acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EV capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4 °C and -80 °C, enhancing the ability to protect EVs while maintaining targeting functionality.

Advances of engineered extracellular vesicles-based therapeutics strategy.

Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-bound vesicles which encapsulate bioactive molecules, such as nucleic acids, proteins, and lipids. They mediate intercellular communication through transporting internally packaged molecules, making them attractive therapeutics carriers. Over the last decades, a significant amount of research has implied the potential of EVs servings as drug delivery vehicles for nuclear acids, proteins, and small molecular drugs. However, several challenges remain unresolved before the clinical application of EV-based therapeutics, including lack of specificity, stability, biodistribution, storage, large-scale manufacturing, and the comprehensive analysis of EV composition. Technical development is essential to overcome these issues and enhance the pre-clinical therapeutic effects. In this review, we summarize the current advancements in EV engineering which demonstrate their therapeutic potential.

Corrigendum: Use of the index of pulmonary vascular disease for predicting longterm outcome of pulmonary arterial hypertension associated with congenital heart disease.

[This corrects the article DOI: 10.3389/fcvm.2023.1212882.].

MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.

Cytomegalovirus retinitis as an adverse immunological effect of pulses of vincristine and dexamethasone in maintenance therapy for childhood acute lymphoblastic leukemia.

We describe a 5-year-old female with acute lymphoblastic leukemia (ALL) who suffered from cytomegalovirus (CMV) retinitis during maintenance therapy consisting of 6-mercaptopurine (6-MP) and methotrexate (MTX) with pulses of vincristine (VCR) and dexamethasone (DEX). Administration of anticytomegaloviral drugs led to a complete regression of active retinitis. Her low CD4 positive T cells and serum immunoglobulin G (IgG) recovered when maintenance therapy was resumed without VCR and DEX. The patient has been in complete remission (CR) for more than 5 months after completion of maintenance therapy without recurrence of CMV retinitis.

Modification of Extracellular Vesicle Surfaces: An Approach for Targeted Drug Delivery.

Extracellular vesicles (EVs) are a promising drug delivery vehicle candidate because of their natural origin and intrinsic function of transporting various molecules between different cells. Several advantages of the EV delivery platform include enhanced permeability and retention effect, efficient interaction with recipient cells, the ability to traverse biological barriers, high biocompatibility, high biodegradability, and low immunogenicity. Furthermore, EV membranes share approximately similar structures and contents to the cell membrane, which allows surface modification of EVs, an approach to enable specific targeting. Enhanced drug accumulation in intended sites and reduced adverse effects of chemotherapeutic drugs are the most prominent effects of targeted drug delivery. In order to improve the targeting ability of EVs, chemical modification and genetic engineering are the most adopted methods to date. Diverse chemical methods are employed to decorate EV surfaces with various ligands such as aptamers, carbohydrates, peptides, vitamins, and antibodies. In this review, we introduce the biogenesis, content, and cellular pathway of natural EVs and further discuss the genetic modification of EVs, and its challenges. Furthermore, we provide a comprehensive deliberation on the various chemical modification methods for improved drug delivery, which are directly related to increasing the therapeutic index.

Erratum: Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo: Erratum.

[This corrects the article DOI: 10.7150/ntno.54879.].

Use of the index of pulmonary vascular disease for predicting long-term outcome of pulmonary arterial hypertension associated with congenital heart disease.

Aims: Limited data exist on risk factors for the long-term outcome of pulmonary arterial hypertension (PAH) associated with congenital heart disease (CHD-PAH). We focused on the index of pulmonary vascular disease (IPVD), an assessment system for pulmonary artery pathology specimens. The IPVD classifies pulmonary vascular lesions into four categories based on severity: (1) no intimal thickening, (2) cellular thickening of the intima, (3) fibrous thickening of the intima, and (4) destruction of the tunica media, with the overall grade expressed as an additive mean of these scores. This study aimed to investigate the relationship between IPVD and the long-term outcome of CHD-PAH. Methods: This retrospective study examined lung pathology images of 764 patients with CHD-PAH aged <20 years whose lung specimens were submitted to the Japanese Research Institute of Pulmonary Vasculature for pulmonary pathological review between 2001 and 2020. Clinical information was collected retrospectively by each attending physician. The primary endpoint was cardiovascular death. Results: The 5-year, 10-year, 15-year, and 20-year cardiovascular death-free survival rates for all patients were 92.0%, 90.4%, 87.3%, and 86.1%, respectively. The group with an IPVD of ≥2.0 had significantly poorer survival than the group with an IPVD <2.0 (P = .037). The Cox proportional hazards model adjusted for the presence of congenital anomaly syndromes associated with pulmonary hypertension, and age at lung biopsy showed similar results (hazard ratio 4.46; 95% confidence interval: 1.45-13.73; P = .009). Conclusions: The IPVD scoring system is useful for predicting the long-term outcome of CHD-PAH. For patients with an IPVD of ≥2.0, treatment strategies, including choosing palliative procedures such as pulmonary artery banding to restrict pulmonary blood flow and postponement of intracardiac repair, should be more carefully considered.

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display.

BACKGROUND: Extracellular vesicles (EVs) are attracting interest as a new class of drug delivery vehicles due to their intrinsic nature of biomolecular transport in the body. We previously demonstrated that EV surface modification with tissue-specific molecules accomplished targeted EV-mediated DNA delivery. METHODS: Here, we describe reliable methods for (i) generating EGFR tumor-targeting EVs via the display of high-affinity monobodies and (ii) in vitro measurement of EV binding using fluorescence and bioluminescence labeling. Monobodies are a well-suited class of small (10 kDa) non-antibody scaffolds derived from the human fibronectin type III (FN3) domain. RESULTS: The recombinant protein consists of the EGFR-targeting monobody fused to the EV-binding domain of lactadherin (C1C2), enabling the monobody displayed on the surface of the EVs. In addition, the use of bioluminescence or fluorescence molecules on the EV surface allows for the assessment of EV binding to the target cells. CONCLUSIONS: In this paper, we describe methods of EV engineering to generate targeted delivery vehicles using monobodies that will have diverse applications to furnish future EV therapeutic development, including qualitative and quantitative in vitro evaluation for their binding capacity.

Engineering Extracellular Vesicles to Target Pancreatic Tissue In Vivo.

Extracellular vesicles (EVs) are naturally released, cell-derived vesicles that mediate intracellular communication, in part, by transferring genetic information and, thus, have the potential to be modified for use as a therapeutic gene or drug delivery vehicle. Advances in EV engineering suggest that directed delivery can be accomplished via surface alterations. Here we assess enriched delivery of engineered EVs displaying an organ targeting peptide specific to the pancreas. We first characterized the size, morphology, and surface markers of engineered EVs that were decorated with a recombinant protein specific to pancreatic ß-cells. This ß-cell-specific recombinant protein consists of the peptide p88 fused to the EV-binding domain of lactadherin (C1C2). These engineered EVs, p88-EVs, specifically bound to pancreatic ß-cells in culture and transferred encapsulated plasmid DNA (pDNA) as early as in 10 min suggesting that the internalization of peptide-bearing EVs is a rapid process. Biodistribution of p88-EVs administrated intravenously into mice showed an altered pattern of EV localization and improved DNA delivery to the pancreas relative to control EVs, as well as an accumulation of targeting EVs to the pancreas using luciferase activity as a readout. These findings demonstrate that systemic administration of engineered EVs can efficiently deliver their cargo as gene carriers to targeted organs in live animals.

DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1.

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

A Comprehensive Review of Cancer MicroRNA Therapeutic Delivery Strategies.

In the field of molecular oncology, microRNAs (miRNAs) and their role in regulating physiological processes and cancer pathogenesis have been a revolutionary discovery over the last decade. It is now considered that miRNA dysregulation influences critical molecular pathways involved in tumor progression, invasion, angiogenesis and metastasis in a wide range of cancer types. Hence, altering miRNA levels in cancer cells has promising potential as a therapeutic intervention, which is discussed in many other articles in this Special Issue. Some of the most significant hurdles in therapeutic miRNA usage are the stability and the delivery system. In this review, we cover a comprehensive update on the challenges and strategies for the development of therapeutic miRNA delivery systems that includes virus-based delivery, non-viral delivery (artificial lipid-based vesicles, polymer-based or chemical structures), and recently emerged extracellular vesicle (EV)-based delivery systems.

Nano-immunoimaging.

Immunoimaging is a rapidly growing field stoked in large part by the intriguing triumphs of immunotherapy. On the heels of immunotherapy's successes, there exists a growing need to evaluate tumor response to therapy particularly immunotherapy, stratify patients into responders vs. non-responders, identify inflammation, and better understand the fundamental roles of immune system components to improve both immunoimaging and immunotherapy. Innovative nanomaterials have begun to provide novel opportunities for immunoimaging, in part due to their sensitivity, modularity, capacity for many potentially varied ligands (high avidity), and potential for multifunctionality/multimodality imaging. This review strives to comprehensively summarize the integration of nanotechnology and immunoimaging, and the field's potential for clinical applications.

Preparation of full-length cDNA libraries: focus on metazoans.

Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

MicroRNA-203 Inversely Correlates with Differentiation Grade, Targets c-MYC, and Functions as a Tumor Suppressor in cSCC.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant recipients. MicroRNAs (miR) are short RNAs that regulate gene expression and cellular functions. Here, we show a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility, and the angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 showed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC as a hub of miR-203-induced transcriptomic changes in squamous cell carcinoma. We identified c-MYC as a direct target of miR-203. Overexpression of c-MYC in rescue experiments reversed miR-203-induced growth arrest in cSCC, which highlights the importance of c-MYC within the miR-203-regulated gene network. Together, miR-203 acts as a tumor suppressor in cSCC, and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide a therapeutic benefit, particularly in poorly differentiated cSCC.

The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression.

Mammalian target of rapamycin (mTOR) signaling is a critical pathway in the biology of acute myeloid leukemia (AML). Proviral integration site for moloney murine leukemia virus (PIM) serine/threonine kinase signaling takes part in various pathways exerting tumorigenic properties. We hypothesized that the combination of a PIM kinase inhibitor with an mTOR inhibitor might have complementary growth-inhibitory effects against AML. The simultaneous inhibition of the PIM kinase by pan-PIM inhibitor AZD1208 and of mTOR by selective mTORC1/2 dual inhibitor AZD2014 exerted anticancer properties in AML cell lines and in cells derived from primary AML samples with or without supportive stromal cell co-culture, leading to suppressed proliferation and increased apoptosis. The combination of AZD1208 and AZD2014 rapidly activated AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells; profoundly inhibited AKT and 4EBP1 activation; and suppressed polysome formation. Inhibition of both mTOR and PIM counteracted induction of heat-shock family proteins, uncovering the master negative regulation of heat shock factor 1 (HSF1), the dominant transcription factor controlling cellular stress responses. The novel combination of the dual mTOR inhibitor and pan-PIM inhibitor synergistically inhibited AML growth by effectively reducing protein synthesis through heat shock factor pathway suppression.

Detection of Mucor velutinosus in a blood culture after autologous peripheral blood stem cell transplantation : a pediatric case report.

Filamentous fungi were detected in the blood culture of a one-year-old boy after autologous peripheral blood stem cell transplantation. The patient was suspected to have aspergillosis and received micafungin. Fungi were isolated on potato dextrose agar medium and incubated at 37℃ for 2-5 days. Grayish, cottony colonies formed. A slide culture showed a spherical sporangium at the tips of the sporangiophores. The fungus could have been a zygomycete. The zygomycete was isolated from three blood cultures. The antifungal drug was changed from micafungin to liposomal amphotericin B, which resulted in an improvement in the patient's symptoms. Growth was observed at 37℃, but not 42℃ in a growth temperature test. Gene sequence analysis identified the fungus as Mucor velutinosus. To the best of our knowledge, this is the first time M. velutinosus has been detected in Japan, and this case is very rare. Zygomycetes are known to be pathogens that cause fungal infections in immunodeficient patients such as those with leukemia. They are difficult to identify by culture and are identified at autopsy in many cases. Therefore, culture examinations should be performed for immunodeficient patients with the consideration of zygomycetes.