Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising ...approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (∼50%; p ≤ 0.02 for interleukin IL-6, tumor necrosis factor TNF-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (∼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (∼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.
Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na
and K
channel clustering ...are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.
Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, ...comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.
The impact of abnormal feeding behaviors reported for children with autism spectrum disorders (ASDs) on their nutritional status is unknown. We compared nutrient intake from food consumed by children ...with and without ASD and examined nutrient deficiency and excess.
Prospective 3-day food records and BMI for children (2-11 years) with ASD participating in the Autism Treatment Network (Arkansas, Cincinnati, Colorado, Pittsburgh, and Rochester) were compared with both the National Health and Nutrition Examination Survey data and a matched subset based on age, gender, family income, and race/ethnicity (N = 252 analyzed food records).
Children with ASD and matched controls consumed similar amounts of nutrients from food. Only children with ASD aged 4 to 8 years consumed significantly less energy, vitamins A and C, and the mineral Zn; and those 9 to 11 years consumed less phosphorous. A greater percentage of children with ASD met recommendations for vitamins K and E. Few children in either group met the recommended intakes for fiber, choline, calcium, vitamin D, vitamin K, and potassium. Specific age groups consumed excessive amounts of sodium, folate, manganese, zinc, vitamin A (retinol), selenium, and copper. No differences were observed in nutritional sufficiency of children given restricted diets. Children aged 2 to 5 years with ASD had more overweight and obesity, and children 5 to 11 years had more underweight.
Children with ASD, like other children in America, consume less than the recommended amounts of certain nutrients from food. Primary care for all children should include nutritional surveillance and attention to BMI.
Background:
In the setting of the COVID pandemic, many patients falling ill with acute respiratory distress syndrome eventually require prone positioning for gas exchange. Traditionally, central ...venous catheters are inserted with patient in the supine or Trendelenburg position. However, when a patient cannot tolerate supine position and the need for central venous access is urgent, catheter placement may be considered with the patient in the prone position.
Case Summary:
A 69-year-old male with rapidly declining respiratory status secondary to COVID pneumonia quickly developed acute respiratory distress syndrome, was rapidly intubated, and then placed in the prone position. Patient could not tolerate the supine position even briefly and required a central venous catheter insertion for continuous renal replacement therapy. We kept the patient in the prone position and successfully inserted a central venous catheter in such position with real-time ultrasound guidance and using micropuncture technique.
Conclusion:
In the setting of the COVID pandemic, many cases of acute respiratory distress syndrome require patients to be prone in order to improve gas exchange. In the most severe situations, these patients would not be able to tolerate rotating back to the supine position but would still require central venous catheter insertion urgently. We demonstrated feasibility of central venous catheter insertion in the prone position in these severely ill patients.
The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that ...coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.
In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 Cas9, and guide RNA gRNA) needed to achieve ...high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these “all-in-one” HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.
Alpha‐1‐anti‐trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy ...increases degradation of mutant, hepatotoxic alpha‐1‐anti‐trypsin (ATZ). We investigated the therapeutic potential of liver‐directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha‐1‐anti‐trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL‐6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha‐1‐anti‐trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.
The authors show that transcription factor EB (TFEB) gene transfer corrects liver disease in alpha‐1‐antitrypsin deficiency, underlying the potential to extend this novel strategy to other human disorders.
Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we ...identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by α-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that α-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that α-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.
Performance assessment and feedback are critical factors in successful medical simulation-based training. The Dynamic Haptic Robotic Trainer (DHRT) allows residents to practice ultrasound-guided ...needle insertions during simulated central venous catheterization (CVC) procedures while providing detailed feedback and assessment. A study was performed to examine the effectiveness of the DHRT in training the important skills of needle tip tracking and aspiration and how these skills impact procedural complications in simulated CVC.
The DHRT data were collected for 163 residents at 2 hospitals for 6 simulated needle insertions. Users were given automated feedback on 5 performance metrics, which measure aspiration rate, arterial punctures, punctures through and through the vein, loss of access to the vein, and successful access to the vein. Aspiration rates and tip tracking rates were analyzed to determine their significance in preventing CVC complications and improving performance.
Tip tracking rates higher than 40% were 2.3 times more likely to result in successful venous access than rates less than 10%. Similarly, aspiration rates higher than 80% were 2.6 times more likely to result in successful venous access than rates less than 10%. Proper tip tracking and aspiration both reduced mechanical complications. Resident performance improved for all metrics except tip tracking.
Proper tip tracking and aspiration both reduced complications and increased the likelihood of success. However, the skill of tip tracking was not effectively learned through practice without feedback. Therefore, ultrasound-guided needle-based procedures, including CVC, can be improved by providing specific feedback to users on their ultrasound usage to track needle insertions.
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