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.
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.
Purpose: Social contact is known to be vital for older adults' mental and physical health but, because communication impairments often co-occur with other types of disability, it is difficult to ...generalize about the relative impact of a communication impairment on the social relationships of older adults. Specific aims of the study were to examine whether the severity of a communication impairment was associated with a range of social measures and to examine the association between these characteristics and psychological well-being. Method: Community-dwelling older adults ranging in age from 65 to 94 were recruited for the study of Communication, Health, Aging, Relationship Types and Support. The sample included 240 participants with communication disorders arising from a variety of etiologies including hearing impairment, voice disorders, head and neck cancer, and neurologic disease, as well as older adults without a communication disorder. Results: Communication impairment was a significant independent predictor for key characteristics of social relationships, including the number of friends in the social network, two types of social support, the frequency of social participation, and social self-efficacy. Communication impairment was also a significant predictor for higher levels of loneliness and depression. In addition, two distinct pathways between communication impairment and psychological well-being were identified, with social self-efficacy and reassurance of worth as mediators. Conclusions: Even after controlling for age, gender, health, and disability, communication impairment is a significant independent predictor for key aspects of the social function of older adults and demonstrates two distinct pathways to loneliness and depression.
The Swallow Disturbance Questionnaire (SDQ) is a screening tool developed to identify patients with Parkinson’s Disease (PD) in need of objective swallowing evaluation. In a previous investigation, ...the SDQ did not predict abnormal airway protection on a videofluoroscopic swallowing study (VFSS). This investigation was undertaken to determine whether SDQ scores were more accurate when a global measure was used. The Dynamic Imaging Grade for Swallowing Toxicity (DIGEST) is a validated measure that provides a safety, efficiency, and total severity grade based on VFSS. A secondary analysis was performed using data from 20 patients with PD who had participated in a standardized VFSS protocol. The study sample was predominantly male (80%) with an average age of 71 years, and an average PD duration of 9 years. Using an established cut-off score, participants were subdivided into those with “normal” (
n
= 10) and “abnormal” SDQ scores (
n
= 10). Recordings were scored using the DIGEST protocol by two blinded raters who also rated overall dysphagia severity from the VFSS. There was good agreement between the two raters on the DIGEST and strong correlations between DIGEST scores and clinician perceptions of dysphagia severity. Higher SDQ scores were associated with poorer Efficiency on the DIGEST but not Safety or Total scores. Consistent with other PD studies, subjective perceptions of dysphagia were poorly predictive of objective findings on VFSS. There is little information about the validity of the DIGEST for rating neurogenic dysphagia. Our study provides preliminary support for the use of the DIGEST in the PD population.
Objectives:
Supracricoid partial laryngectomy (SCPL) is an oncologically sound alternative to total laryngectomy that results in long-term alterations in vocal function. Little is known about ...long-term improvements in dysphonia and the mechanism of vocal recovery is unclear due to the lack of a standardized rating tool: The Pattern, Degree, and Vibration (PDV) Scale.
Methods:
Data from 24 individuals were compared over 3 post-operative timepoints after SCPL: 3 to 11, 12 to 35, and over 35 months. Voice outcomes were assessed using the Voice Handicap Index (VHI) and the GRBAS scale. Laryngeal exams were deidentified and rated using a novel rating scheme developed using literature review and consensus panel discussions.
Results:
There were significant improvements in VHI scores, Grade, and Strain over time. There was an increase in the Degree of Closure and a decline in Mucosal Vibration across timepoints. Pattern of Movement (P) was associated with dysphonia Grade. Better Degree of Closure (D) was associated with lower VHI scores and better Grade and Roughness. Mucosal Vibration (V) was associated with reduced Breathiness and Strain but variable Roughness. Age, T-stage, radiation treatment, surgery type, and time to feeding-tube removal were also associated with voicing characteristics.
Conclusions:
There is evidence of improvement in several voice parameters over time after the first post-operative year. Various subcomponents of the new PDV rating scale were associated with voice outcomes. Its utility for research and clinical practice merits further investigation.
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.
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.
Adenoviruses are robust gene delivery vectors in vivo, but are limited by their propensity to provoke strong innate and adaptive responses. Previous work has demonstrated that polyethylene glycol ...(PEG) modification of adenovirus can protect the vectors from preexisting and adaptive immune responses by reducing protein–protein interactions. To test whether PEGylation can reduce innate immune responses to adenovirus by reducing their interactions with immune cells, first-generation (FG-Ad) and helper-dependent (HD-Ad) Ad5 vectors were PEGylated with SPA-PEG and tested in vitro and in vivo. We demonstrate that increasing PEGylation ablated in vitro transduction, but surprisingly had no negative effect on the level or distribution of in vivo gene delivery. This poor in vitro transduction could be rescued in part by physically forcing the PEGylated vectors onto cells, suggesting that physiological forces in vivo may enable transduction via heparin sulfate proteoglycan and integrin interactions. While transduction remained the same as for unmodified vectors, the PEGylated vectors reduced innate IL-6 responses by 70 and 50% in vivo for FG-Ad and HD-Ad. These reduced innate responses paralleled similar reductions in vector uptake by macrophages in vitro and Kupffer cells in vivo. These data suggest that PEGylation of Ad vectors can reduce innate immune responses without reducing transduction in vivo. These data also suggest that nonspecific vector uptake by macrophages and Kupffer cells may be critically involved in the initial activation of innate immune responses.
•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.
PDF
