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Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl- transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with ∼20% wild-type cells generated ∼70% the transepithelial Cl- current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl- current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl- flow, thereby reducing net transepithelial Cl- transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl- transport because transepithelial Cl- flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane. [ABSTRACT FROM AUTHOR]

Contrary to the pervasive negative stereotypes of human aging, emotional functions may improve with advancing age. However, the brain mechanisms underlying changes in emotional function over age remain unknown. Here, we demonstrate that emotional stability improves linearly over seven decades (12-79 years) of the human lifespan. We used both functional magnetic resonance imaging and event-related potential recording to examine the neural basis of this improvement. With these multimodal techniques, we show that better stability is predicted by a shift toward greater medial prefrontal control over negative emotional input associated with increased activity later in the processing sequence (beyond 200 ms after stimulus) and less control over positive input, related to a decrease in early activity (within 150 ms). This shift was independent from gray matter loss, indexed by structural magnetic resonance data. We propose an integrative model in which accumulated life experience and the motivation for meaning over acquisition in older age contribute to plasticity of medial prefrontal systems, achieving a greater selective control over emotional functions. [ABSTRACT FROM AUTHOR]

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. Author summary: Adenoviruses can be engineered to create non-replicating vectors that are useful for gene therapy or vaccination. Many Ad gene therapy vectors can specifically bind coagulation factors from the blood, and coagulation factors can influence the ability of intravenously-injected Ad vectors to deliver their genes to the liver. We show that delivery of Ad vectors to the mouse liver is more strongly dependent on coagulation factors for some Ad vectors than for others. We also demonstrate novel interactions between Ad and prothrombin, which is the most abundant coagulation factor in blood. We find that Ad vectors differ in their ability to bind to human and mouse coagulation factors, which is important because mice are a common preclinical model for studies with Ad vectors. Our studies define the interaction of Ad gene therapy vectors with coagulation factors, and the results suggest ways to design gene therapy vectors that have increased or decreased ability to target the liver after intravenous administration. [ABSTRACT FROM AUTHOR]

The viscosity of a supercritical electrolyte solution is measured for the first time using a modified quartz crystal microbalance, and it is shown that ionic solvation leads to a significant structuring of the solvent and an appreciable increase in solution viscosity. Voltammetric investigations in the electrolyte solutions are used to confirm the magnitude of the viscosity changes, and these account for the appreciably lower than expected peak currents. [ABSTRACT FROM AUTHOR]

MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function. [ABSTRACT FROM AUTHOR]

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase-dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism. [ABSTRACT FROM AUTHOR]

Prevalence rates of attention-deficit/hyperactivity disorder (ADHD) differ with geographical areas varying in sunlight intensity. Sun- or daylight reaching the retina establishes entrainment of the circadian clock to daylight. Changes herein, hence, alterations in clock alignment, could be reflected indirectly in inattention via sleep duration. We here studied (1) annual variation in inattention at treatment initiation; (2) annual variation in response to ADHD treatment [methylphenidate (MPH)] by day of treatment initiation; and (3) dose dependence. We predicted least baseline inattention during a period of high sunlight intensity implying more room for improvement (i.e., a better treatment response) when sunlight intensity is low. These hypotheses were not confirmed. High-dose treated patients, however, had significantly better attention after treatment than low-dosed treated patients, only when treated in the period from winter to summer solstice. Change in solar irradiance (SI) during low-dosed treatment period was negatively related to attentional improvement. The above described findings were primarily found in inattention ratings and replicated in omission errors on a continuous performance task. Daylight and inattention have been proposed to be related via mediation of the circadian system. One mechanism of MPH may be to enhance sensitivity to the diurnal entrainment to sunlight and the question can be raised whether appropriate lighting could potentiate the effects of stimulants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Functional somatic symptoms (FSS) emerge when the stress system is activated in response to physical or emotional stress that is either chronic or especially intense. In such cases, the heightened state of physiological arousal and motor activation can be measured through biological markers. Our team have integrated the use of biological markers of body state – respiratory rate, heart rate (HR) and heart rate variability (HRV) measurements – as a way of helping families to understand how physical symptoms can signal activation of the body's stress systems. This study measured respiratory rates, HR and HRV in children and adolescents with FSS (and healthy controls) during baseline assessment to determine whether these biological markers were effective at differentiating patients with FSS. The study also implemented a biofeedback intervention during the assessment to determine whether patients with FSS were able to slow their respiratory rates and increase HRV. Patients with FSS had faster respiratory rates, faster HR, and lower HRV, suggesting activation of the autonomic nervous system coupled with activation of the respiratory motor system. Like controls, patients were able to slow their respiratory rates, but in contrast to controls, they were unable to increase their HRV. Our findings suggest that patients with FSS present in a state of physiological activation and struggle to regulate their body state. Patients with FSS are likely to need ongoing training and practice to regulate body state coupled with interventions that target regulatory capacity across multiple systems. [ABSTRACT FROM AUTHOR]

Abstract Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches. [ABSTRACT FROM AUTHOR]

The aims of this randomized, double-blind, three-arm, single-dose study were to demonstrate pharmacokinetic (PK) equivalence of the adalimumab biosimilar M923 (hereafter referred to as "M923") to each of 2 reference products, and to assess M923's safety and immunogenicity. Primary PK endpoints were maximum observed concentration (Cmax), area under the curve (AUC) from time 0 extrapolated to infinity (AUC0-inf), and AUC from time 0 to 336 hours (AUC0-336). Secondary endpoints included safety and immunogenicity assessments. Healthy subjects were randomized 1:1:1 to receive a 40-mg dose of M923 (n = 107); adalimumab US Humira (n = 105), hereafter referred to as "US Humira"; or adalimumab EU Humira (n = 103), hereafter referred to as "EU Humira." PK equivalence was demonstrated for all primary PK endpoints. Geometric least squares means ratios (GMRs) for Cmax, AUC0-inf, and AUC0-336 were 99.4, 100.9, and 100.5, respectively, between the M923 and EU Humira arms and 102.6, 104.2, and 102.9 between the M923 and US Humira arms. The 90% confidence intervals of the GMRs for all PK endpoints were within prespecified confidence bounds of 80%-125%. Adverse event rates were similar across the M923 (47.7%), US Humira (50.9%), and EU Humira (53.3%) arms and were generally mild (73.7%) or moderate (22.0%). The proportion of subjects with a confirmed antidrug antibody (ADA) response was similar across study arms. This study demonstrated bioequivalent PK among M923, US Humira, and EU Humira and demonstrated that the PK parameters were consistent with similar safety and tolerability profile and ADA response rates. [ABSTRACT FROM AUTHOR]

• N-terminal eGFP-tagging of rabbit apoA-I decreases protein expression and secretion. • N-terminal eGFP-tagging of rabbit apoA-I eliminates cholesterol efflux activity. [ABSTRACT FROM AUTHOR]

Children and adolescents with conversion disorders and their families are challenging to understand and challenging to treat. This study examined whether self-report instruments are a useful adjunct to family and individual assessment. A total of 57 children and adolescents diagnosed with conversion disorders and 57 sex- aged-matched controls, along with their parents, completed the Strength and Difficulties Questionnaire, Child Behaviour Checklist, Depression Anxiety and Stress Scales, Somatic and Psychological Health Report ( SPHERE), Brief Risk-Resilience Index for Screening, and the NEO-Five-Factor Inventory. The conversion disorder and control groups were compared on each questionnaire, and classified participants as 'cases' or not according to established clinical thresholds for each questionnaire. A discriminant analysis was used to examine which components of the SDQ, CBCL, DASS, SPHERE, BRISC, and NEO- FFI emerged as most useful in identifying the clinical characteristics of children with conversion disorders. The role of attachment strategy with regards to the utility of self-report measures was also examined. The SPHERE was the only self-report that our patient cohort were able to utilise to communicate their somatic and emotional discomfort and distress. Our results suggest that children and adolescents presenting with functional somatic symptoms represent a distinctive clinical population for which the 'psychological' focus of most currently available questionnaires is not useful for identifying the presence of somatic illness or of comorbid anxiety and depression, and that these measures - with the exception of the SPHERE - are not a useful adjunct to a family-based assessment. Therapeutic conversations that utilise language that asks about the state of the body - the body's response to stress, illness, pain, and injury - facilitate engagement and serve as the cornerstone of treatment. [ABSTRACT FROM AUTHOR]

An abstract of the article "High Efficiency Hepatic Transduction with Minimal Toxicity Following “Hydrodynamic” Injection of HDAd into Nonhuman Primates," by Nicola Brunetti-Pierri and colleagues is presented.

Current protocols for hematopoietic stem/progenitor cell (HSPC) gene therapy, involving the transplantation of ex vivo genetically modified HSPCs are complex and not without risk for the patient. We developed a new approach for in vivo HSPC transduction that does not require myeloablation and transplantation. It involves subcutaneous injections of granulocyte-colony-stimulating factor/AMD3100 to mobilize HSPCs from the bone marrow (BM) into the peripheral blood stream and the IV injection of an integrating, helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is uniformly expressed on HSPCs. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSPCs transduced in the periphery home back to the BM where they stably express the transgene. In hCD46 transgenic mice, we showed that our in vivo HSPC transduction approach allows for the stable transduction of primitive HSPCs. Twenty weeks after in vivo transduction, green fluorescent protein (GFP) marking inBMHSPCs(Lin2Sca11Kit2 cells) in most of the mice was in the range of 5% to 10%. The percentage of GFP-expressing primitive HSPCs capable of forming multilineage progenitor colonies (colony-forming units [CFUs]) increased from 4% of all CFUs at week 4 to 16% at week 12, indicating transduction and expansion of long-term surviving HSPCs. Our approach was well tolerated, did not result in significant transduction of nonhematopoietic tissues, and was not associated with genotoxicty. The ability to stably genetically modify HSPCs without the need of myeloablative conditioning is relevant for a broader clinical application of gene therapy. [ABSTRACT FROM AUTHOR]

Objective: To determine whether EEG occipital alpha and frontal alpha asymmetry (FAA) distinguishes outpatients with major depression (MDD) from controls, predicts antidepressant treatment outcome, and to explore the role of gender. Methods: In the international Study to Predict Optimized Treatment in Depression (iSPOT-D), a multi-center, randomized, prospective open-label trial, 1008 MDD participants were randomized to escitalopram, sertraline or venlafaxine-extended release. The study also recruited 336 healthy controls. Treatment response was established after eight weeks and resting EEG was measured at baseline (two minutes eyes open and eyes closed). Results: No differences in EEG alpha for occipital and frontal cortex, or for FAA, were found in MDD participants compared to controls. Alpha in the occipital and frontal cortex was not associated with treatment outcome. However, a gender and drug-class interaction effect was found for FAA. Relatively greater right frontal alpha (less cortical activity) in women only was associated with a favorable response to the Selective Serotonin Reuptake Inhibitors escitalopram and sertraline. No such effect was found for venlafaxine-extended release. Conclusions: FAA does not differentiate between MDD and controls, but is associated with antidepressant treatment response and remission in a gender and drug-class specific manner. Significance: Future studies investigating EEG alpha measures in depression should a-priori stratify by gender. [ABSTRACT FROM AUTHOR]