Probing aspects of extracellular vesicle associated AAV allows increased vector yield and insight into its transduction and immune-evasive properties.

Extracellular vesicle-associated adeno-associated virus vectors (EV-AAVs) are generated during production in 293 cells. EV-AAV provides desirable gene delivery traits such as greater resistance to antibody neutralization and increased transduction of organs compared with conventional AAV. Despite these promising data, better characterization of EV-AAV is needed.

An and efficacy evaluation of gene therapy candidate SBT101 in mouse models of adrenomyeloneuropathy and in NHPs.

Adrenomyeloneuropathy is a progressive neurodegenerative disease caused by pathogenic variants in the gene, resulting in very-long-chain fatty acid (VLCFA) accumulation that leads to dying-back axonopathy. Our candidate gene therapy, SBT101 (AAV9-human [h]), aims to ameliorate pathology by delivering functional copies of h to the spinal cord. Transduced cells produce functional ABCD1 protein, thereby repairing the underlying biochemical defect.

Expression-based selection identifies a microglia-tropic AAV capsid for direct and CSF routes of administration in mice.

Microglia are critical innate immune cells of the brain. targeting of microglia using gene-delivery systems is crucial for studying brain physiology and developing gene therapies for neurodegenerative diseases and other brain disorders such as NeuroAIDS. Historically, microglia have been extremely resistant to transduction by viral vectors, including adeno-associated virus (AAV) vectors.

Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV.

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes.

selection in non-human primates identifies superior AAV capsids for on-target CSF delivery to spinal cord.

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal delivery of AAV vectors into the cerebral spinal fluid (CSF) can avoid many of the issues of systemic delivery, although achieving broad distribution of the vector and transgene expression throughout the spinal cord is challenging and vector entry to the periphery occurs, sometimes initiating hepatotoxicity. Here we performed two rounds of biopanning in non-human primates (NHPs) with an AAV9 peptide display library injected intrathecally and performed insert sequencing on DNA isolated from either whole tissue (conventional selection), isolated nuclei, or nuclei from transgene-expressing cells.

An Engineered Adeno-Associated Virus Capsid Mediates Efficient Transduction of Pericytes and Smooth Muscle Cells of the Brain Vasculature.

Neurodegeneration and cerebrovascular disease share an underlying microvascular dysfunction that may be remedied by selective transgene delivery. To date, limited options exist in which cellular components of the brain vasculature can be effectively targeted by viral vector therapeutics. In this study, we characterize the first engineered adeno-associated virus (AAV) capsid mediating high transduction of cerebral vascular pericytes and smooth muscle cells (SMCs).

Baveno VI and VII criteria are not suitable for screening for large varices or clinically significant portal hypertension in patients with hepatocellular carcinoma.

Background: Baveno VI and VII criteria are used in patients with cirrhosis to rule out large size oesophageal varices (EV) and rule in/out clinically significant portal hypertension (CSPH).

Aim: To evaluate their diagnostic performance in these patients.

Methods: We retrospectively included all patients with Child-Pugh A cirrhosis and HCC who had endoscopy, liver stiffness measurement (LSM) and platelet count within 6 months.

Neutralizing Antibody Evasion and Transduction with Purified Extracellular Vesicle-Enveloped Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) is classified as a nonenveloped DNA virus. However, several years ago, we discovered that in media of packaging cells producing recombinant AAV vectors, AAV capsids can associate with the interior and surface of extracellular vesicles (EVs), sometimes referred to as exosomes. Since then, we and others have demonstrated that exosome-enveloped AAV, exo-AAV, can enhance transduction as well as evade neutralizing antibodies.

The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration.

Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection.

Diagnosing Acute Heart Failure in the Pediatric Emergency Department Using Point-of-Care Ultrasound.

Background: Acute heart failure (AHF) in children is associated with significant disease burden with high rates of morbidity, mortality, and resource utilization. These children often present to the emergency department with clinical features that mimic common childhood illnesses. Cardiac point-of-care ultrasound (POCUS) can be an effective tool for rapidly identifying abnormal cardiac function.

The effect of point-of-care ultrasound on length of stay in the emergency department in children with neck swelling.

Background: Ultrasound is the imaging modality of choice in children presenting to the emergency department (ED) with soft tissue neck swelling. Point of care ultrasound (POCUS) has good accuracy when compared to comprehensive radiology department ultrasound (RADUS). POCUS could potentially improve ED length of stay (LOS) by improving efficiency.

Improving Point-of-Care Ultrasound Documentation and Billing Accuracy in a Pediatric Emergency Department.

Objective: The performance and interpretation of point-of-care ultrasound (POCUS) should be documented appropriately in the electronic medical record (EMR) with correct billing codes assigned. We aimed to improve complete POCUS documentation from 62% to 80% and improve correct POCUS billing codes to 95% or higher through the implementation of a quality improvement initiative.

Methods: We collected POCUS documentation and billing data from the EMR.

Parotitis on Ultrasound: The Pomegranate Sign.

The differential diagnosis for facial swelling is broad and can be a diagnostic challenge in the pediatric emergency department. We describe the first pediatric case of acute parotitis with sialolithiasis where the diagnosis was facilitated by point-of-care ultrasound.

Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons.

Fragile X syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5'-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes.

Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids.

Intrathecal Adeno-Associated Viral Vector-Mediated Gene Delivery for Adrenomyeloneuropathy.

Mutations in the gene encoding the peroxisomal ATP-binding cassette transporter () cause elevations in very long-chain fatty acids (VLCFAs) and the neurodegenerative disease adrenoleukodystrophy (ALD). In most adults, this manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN). A challenge in virus-based gene therapy in AMN is how to achieve functional gene correction to the entire spinal cord while minimizing leakage into the systemic circulation, which could contribute to toxicity.

Attitudes towards three ultrasound-guided vascular access techniques in a paediatric emergency department.

Background: although nurses often place peripheral intravenous (IV) catheters, little is known about their perceptions regarding use of ultrasound guidance.

Aim: the authors aimed to determine paediatric emergency department nurses' attitudes toward ultrasound-guided IV access techniques and assessed practice change after training.

Method: In a cross-sectional study of nurses, they had didactic and hands-on practice sessions, using three short-axis ultrasound-guided IV access techniques on gel models.

Diagnosis of a Peritonsillar Abscess by Transcutaneous Point-of-Care Ultrasound in the Pediatric Emergency Department.

We report a case of a pediatric patient with an initial diagnosis of peritonsillar cellulitis made by otolaryngology. The findings from a subsequent transcutaneous point-of-care ultrasound by a pediatric emergency physician directly affected the decision to perform needle aspiration. Sonographic characteristics of a peritonsillar abscess may be helpful in the prompt diagnosis of peritonsillar abscess.

Avoiding Computed Tomography Scans By Using Point-Of-Care Ultrasound When Evaluating Suspected Pediatric Renal Colic.

Background: Although renal colic in children in the United States remains relatively uncommon compared to in adults, its incidence has nearly doubled from 1999 to 2008. Noncontrast computed tomography (CT) is the current standard for the evaluation of suspected renal colic, given its high sensitivity and specificity. However, the greater lifetime risk of radiation-induced cancer from CT in pediatric patients has led to efforts to minimize radiation exposure.

One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection.

Background: Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH) probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance.

Scratching the surface: a review of skin and soft tissue infections in children.

Purpose Of Review: We present data from recently conducted research on the diagnosis and management of skin and soft tissue infections (SSTIs) in children.

Recent Findings: Current research in the area of SSTIs (cellulitis and abscess) has focused on the use of ultrasound, risk factors associated with bacteremia, antibiotic choice, and incision and drainage (I&D) practices. When clinical examination is equivocal at distinguishing abscess from cellulitis, ultrasound can aid in the diagnosis and alter management.

Fat-Dachsous signaling coordinates cartilage differentiation and polarity during craniofacial development.

Organogenesis requires coordinated regulation of cellular differentiation and morphogenesis. Cartilage cells in the vertebrate skeleton form polarized stacks, which drive the elongation and shaping of skeletal primordia. Here we show that an atypical cadherin, Fat3, and its partner Dachsous-2 (Dchs2), control polarized cell-cell intercalation of cartilage precursors during craniofacial development.