Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration.

The development of liver-based adeno-associated virus (AAV) gene therapies is facing concerns about limited efficiency and durability of transgene expression. We evaluated nonhuman primates following intravenous dosing of AAV8 and AAVrh10 vectors for over 2 years to better define the mechanism(s) of transduction that affect performance. High transduction of non-immunogenic transgenes was achieved, although expression declined over the first 90 days to reach a lower but stable steady state. More than 10% of hepatocytes contained single nuclear domains of vector DNA that persisted despite the loss of transgene expression. Greater reductions in vector DNA and RNA were observed with immunogenic transgenes. Genomic integration of vector sequences, including complex concatemeric structures, were detected in 1 out of 100 cells at broadly distributed loci that were not in proximity to genes associated with hepatocellular carcinoma. Our studies suggest that AAV-mediated transgene expression in primate hepatocytes occurs in two phases: high but short-lived expression from episomal genomes, followed by much lower but stable expression, likely from integrated vectors.

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake.

BACKGROUND: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. RESULTS: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. CONCLUSION: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

Acute Sleep Loss Upregulates the Synaptic Scaffolding Protein, Homer1a, in Non-canonical Sleep/Wake Brain Regions, Claustrum, Piriform and Cingulate Cortices.

Homer proteins are a component of the post-synaptic density of neurons that are necessary for the maintenance and consolidation of behavioral state. The dominant negative protein homer1a is rapidly increased by neuronal activity and sleep loss. Homer1a knockout mice with globally absent homer1a have reduced ability to sustain wakefulness during the active period. It is not known whether homer1a is required globally or in very specific brain regions or neurons for its role in maintaining wake. In this study, we examined the expression of homer1a, an immediate early gene involved in intracellular signaling cascades, in mice subjected to extended wakefulness. We found that mice displayed increased expression of homer1a in the claustrum, a brain region thought to be involved in consciousness, as well as the cingulate and piriform cortices compared to non-sleep deprived mice. In situ hybridization (ISH) studies also indicate that homer1a is not induced in the known wake promoting regions with sleep deprivation, but is instead upregulated primarily in the claustrum and piriform cortex. Examination of homer1a expression levels with recovery sleep after sleep deprivation indicate that baseline homer1a expression levels were restored. Further, we have identified that homer1a is upregulated in excitatory neurons of the claustrum suggesting that homer1a promotes wakefulness through activating excitatory neurons. This work identifies regions previously unknown to be involved in sleep regulation that respond to acute sleep deprivation or enhanced waking.

Reduction of the molecular chaperone binding immunoglobulin protein (BiP) accentuates the effect of aging on sleep-wake behavior.

Sleep and wake quality, quantity, and architecture become modified with aging. Sleep and wake quality decline coinciding with increased fragmentation of both states across aging. We have previously shown that this age-related decline in sleep-wake quality is associated with increased endoplasmic reticular (ER) stress and decreased expression of the major ER chaperone binding immunoglobulin protein (BiP). BiP, also known as glucose-regulated protein 78, plays a key role in controlling the cellular response to ER stress, acting as a regulator of a protein homeostatic signaling pathway known as the unfolded protein response. Induction of BiP during cellular stress is part of an adaptive prosurvival mechanism. Here, using mice heterozygous for BiP, we investigated the effect of reduced BiP expression on sleep-wake behavior across aging; complete knockdown of BiP is embryonic lethal. We report that BiP heterozygosity accentuates the aging sleep-wake phenotype. Sleep and wake fragmentation was more pronounced in the BiP heterozygotes across the 3 ages examined. In mice lacking 1 functional copy of BiP, we observed an age-related significant reduction in wake bout duration and increase in wake bout numbers during the active period, as well as an increase in non rapid eye movement and rapid eye movement bout numbers accompanied by reduced bout durations of both non rapid eye movement and rapid eye movement during the sleep period. In addition, we observed increased ER stress in orexin neurons and occurrence of aggregates immunopositive for orexin at the terminals and projections of orexin neurons in the middle-aged BiP heterozygotes. Taken together, our data indicate that a reduction in the molecular chaperone BiP impacts sleep architecture across aging and that orexin processing is likely to be affected.

Tongue fat infiltration in obese versus lean Zucker rats.

STUDY OBJECTIVES: Obesity is the most important risk factor for obstructive sleep apnea (OSA), and the effects of obesity may be mediated by tongue fat. Our objective was to examine the effects of obesity on upper airway structures in obese (OBZ) and non-obese (NBZ) Zucker rats. DESIGN: Animal study. SETTING: Academic Medical Center. PARTICIPANTS: OBZ (638.2 ± 39 g; 14.9 ± 1.1 w) and age-matched NBZ Zucker (442.6 ± 37 g, 15.1 ± 1.5 w) rats. INTERVENTIONS: TONGUE FAT AND VOLUME AND WERE ASSESSED USING: in vivo magnetic resonance spectroscopy (MRS), magnetic resonance imaging including Dixon imaging for tongue fat volume, ex vivo biochemistry (fat quantification; triglyceride (mg)/tissue (g), and histology (Oil Red O stain). MEASUREMENTS AND RESULTS: MRS: overall OBZ tongue fat/water ratio was 2.9 times greater than NBZ (P < 0.002) with the anterior OBZ tongue up to 3.3 times greater than NBZ (P < 0.002). Biochemistry: Triglyceride (TG) in the tongue was 4.4 times greater in OBZ versus NBZ (P < 0.0006). TG was greater in OBZ tongue (3.57 ± 1.7 mg/g) than OBZ masseter muscle (0.28 ± 0.1; P < 0.0001) but tongue and masseter TG were not different in NBZ rats (0.82 ± 0.3 versus 0.28 ± 0.1 mg/g, P = 0.67). Dixon fat volume was significantly increased in OBZ (56 ± 15 mm3) versus NBZ (34 ± 5 mm3, P < 0.004). Histology demonstrated a greater degree of intracellular muscle fat and extramuscular fat infiltration in OBZ versus NBZ rats. CONCLUSIONS: Genetically obese rats had a large degree of fat infiltration in the tongue compared to both skeletal muscle and tongue tissues of the non-obese age-matched littermates. The significant fat increase and sequestration in the obese tongue may play a role in altered tongue neuromuscular function, tongue stiffness or metabolic function.

Aging and sleep deprivation induce the unfolded protein response in the pancreas: implications for metabolism.

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging.

Dietary therapy mitigates persistent wake deficits caused by mild traumatic brain injury.

Sleep disorders are highly prevalent in patients with traumatic brain injury (TBI) and can significantly impair cognitive rehabilitation. No proven therapies exist to mitigate the neurocognitive consequences of TBI. We show that mild brain injury in mice causes a persistent inability to maintain wakefulness and decreases orexin neuron activation during wakefulness. We gave mice a dietary supplement of branched-chain amino acids (BCAAs), precursors for de novo glutamate synthesis in the brain. BCAA therapy reinstated activation of orexin neurons and improved wake deficits in mice with mild brain injury. Our data suggest that dietary BCAA intervention, acting in part through orexin, can ameliorate injury-induced sleep disturbances and may facilitate cognitive rehabilitation after brain injury.

The development of a novel dry powder inhaler.

A novel active and multi-dose dry powder inhaler (DPI) was developed and evaluated to deliver a small quantity (100-500 µg) of pure drug without any excipient. This dry powder inhaler utilized two compressed air flows to dispense and deliver drug powder: the primary flow aerosolizes the drug powder from its pocket and the secondary flow further disperses the aerosol. In vitro tests by Anderson Cascade Impactor (ACI) indicated that the fine particle fraction (FPF) (<4.7 µm) of drug delivery could reach over a range of 50-70% (w/w). Emitted dose tests showed that delivery efficiency was above 85% and its relative standard deviation (RSD) was under 10%. Confocal microscopy was used to confirm the deposition of fluorescently labeled spray-dried powder in rabbit lungs. Also, a chromatographic method was used to quantify drug deposition. The results of animal tests showed that 57% of aerosol deposited in the rabbit lung and 24% deposited in its trachea. All the results implied that this novel active dry powder inhaler could efficiently deliver a small quantity of fine drug particles into the lung with quite high fine particle fraction.

Endoplasmic reticulum stress in wake-active neurons progresses with aging.

Fragmentation of wakefulness and sleep are expected outcomes of advanced aging. We hypothesize that wake neurons develop endoplasmic reticulum dyshomeostasis with aging, in parallel with impaired wakefulness. In this series of experiments, we sought to more fully characterize age-related changes in wakefulness and then, in relevant wake neuronal populations, explore functionality and endoplasmic reticulum homeostasis. We report that old mice show greater sleep/wake transitions in the active period with markedly shortened wake periods, shortened latencies to sleep, and less wake time in the subjective day in response to a novel social encounter. Consistent with sleep/wake instability and reduced social encounter wakefulness, orexinergic and noradrenergic wake neurons in aged mice show reduced c-fos response to wakefulness and endoplasmic reticulum dyshomeostasis with increased nuclear translocation of CHOP and GADD34. We have identified an age-related unfolded protein response injury to and dysfunction of wake neurons. It is anticipated that these changes contribute to sleep/wake fragmentation and cognitive impairment in aging.

SHP-2 phosphatase activity is required for PECAM-1-dependent cell motility.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in endothelial cell motility during angiogenesis. Although there is evidence that SHP-2 plays a role in PECAM-1-dependent cell motility, the molecular basis of the activity of SHP-2 in this process has not been defined. To investigate the requirement of SHP-2 in PECAM-1-dependent cell motility, studies were done in which various constructs of SHP-2 were expressed in cell transfectants expressing PECAM-1. We observed that the levels of PECAM-1 tyrosine phosphorylation and SHP-2 association with PECAM-1 were significantly increased in cells expressing a phosphatase-inactive SHP-2 mutant, suggesting that the level of PECAM-1 tyrosine phosphorylation, and thus SHP-2 binding are regulated in part by bound, catalytically active SHP-2. We subsequently found that expression of PECAM-1 stimulated wound-induced migration and the formation of filopodia (a morphological feature of motile cells). These activities were associated with increased mitogen-activated protein kinase (MAPK) activation and the dephosphorylation of paxillin (an event implicated in the activation of MAPK). The phosphatase-inactive SHP-2 mutant, however, suppressed these PECAM-1-dependent phenomena, whereas the activity of PECAM-1 expressing cells was not altered by expression of wild-type SHP-2 or SHP-2 in which the scaffold/adaptor function had been disabled. Pharmacological inhibition of SHP-2 phosphatase activity also suppressed PECAM-1-dependent motility. Furthermore, PECAM-1 expression also stimulates tube formation, but none of the SHP-2 constructs affected this process. These findings therefore suggest a model for the involvement of SHP-2 in PECAM-1-dependent motility in which SHP-2, recruited by its interaction with PECAM-1, targets paxillin to ultimately activate the MAPK pathway and downstream events required for cell motility.

Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.

Platelet endothelial cell adhesion molecule (PECAM)-1 has been previously implicated in endothelial cell migration; additionally, anti-PECAM-1 antibodies have been shown to inhibit in vivo angiogenesis. Studies were therefore performed with PECAM-1-null mice to further define the involvement of PECAM-1 in blood vessel formation. Vascularization of subcutaneous Matrigel implants as well as tumor angiogenesis were both inhibited in PECAM-1-null mice. Reciprocal bone marrow transplants that involved both wild-type and PECAM-1-deficient mice revealed that the impaired angiogenic response resulted from a loss of endothelial, but not leukocyte, PECAM-1. In vitro wound migration and single-cell motility by PECAM-1-null endothelial cells were also compromised. In addition, filopodia formation, a feature of motile cells, was inhibited in PECAM-1-null endothelial cells as well as in human endothelial cells treated with either anti-PECAM-1 antibody or PECAM-1 siRNA. Furthermore, the expression of PECAM-1 promoted filopodia formation and increased the protein expression levels of Cdc42, a Rho GTPase that is known to promote the formation of filopodia. In the developing retinal vasculature, numerous, long filamentous filopodia, emanating from endothelial cells at the tips of angiogenic sprouts, were observed in wild-type animals, but to a lesser extent in the PECAM-1-null mice. Together, these data further establish the involvement of endothelial PECAM-1 in angiogenesis and suggest that, in vivo, PECAM-1 may stimulate endothelial cell motility by promoting the formation of filopodia.

Modeling and simulation of liquid-solid circulating fluidized bed ion exchange system for continuous protein recovery.

Liquid-solid circulating fluidized bed (LSCFB) is an integrated two-column (downcomer and riser) system which can accommodate two separate processes (adsorption and desorption) in the same unit with continuous circulation of the solid particles between the two columns. In this study, a mathematical model based on the assumption of homogeneous fluidization was developed considering hydrodynamics, adsorption-desorption kinetics and liquid-solid mass transfer. The simulation results showed good agreement with the available experimental results for continuous protein recovery. A parametric sensitivity study was performed to better understand the influence of different operating parameters on the BSA adsorption and desorption capacity of the system. The model developed can easily be extended to other applications of LSCFB.

Multiobjective optimization of the operation of a liquid-solid circulating fluidized bed ion-exchange system for continuous protein recovery.

Like most real-life processes, the operation of liquid-solid circulating fluidized bed (LSCFB) system for continuous protein recovery is associated with several objectives such as maximization of production rate and recovery of protein, and minimization of amount solid ion-exchange resin requirement, all of which need to be optimized simultaneously. In this article, multiobjective optimization of a LSCFB system for continuous protein recovery was carried out using an experimentally validated mathematical model to find the scope for further improvements in its operation. Elitist non-dominated sorting genetic algorithm with its jumping gene adaptation was used to solve a number of bi- and tri-objective function optimization problems. The optimization resulted in Pareto-optimal solution, which provides a broad range of non-dominated solutions due to conflicting behavior of the operating parameters on the system performance indicators. Significant improvements were achieved, for example, the production rate at optimal operation increased by 33%, using 11% less solid compared to reported experimental results for the same recovery level. The effects of operating variables on the optimal solutions are discussed in detail. The multiobjective optimization study reported here can be easily extended for the improvement of LSCFB system for other applications.

Detachment of multi species biofilm in circulating fluidized bed bioreactor.

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.

Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis.

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.

Continuous protein recovery from whey using liquid-solid circulating fluidized bed ion-exchange extraction.

A liquid-solid circulating fluidized bed (LSCFB) continuous ion-exchange extraction system has been investigated for total protein recovery from whey solutions under various operating conditions. The effectiveness of a dynamic seal was evaluated between the riser and the downcomer, and the best conditions for the establishment of this seal were established. Start-up studies indicated that the system is robust and stable. Under optimal conditions, a productivity of 8.2 g of total protein removed per hour per kilogram of resin was achieved with a protein removal efficiency of 78.4%. However, higher overall protein recovery of up to 90% was also achieved under other conditions, with lower protein concentration in the effluent and a lower overall productivity.

Gene expression of MMP8 and MMP13 during embryonic development of bone and cartilage in the rat mandible and hind limb.

Matrix metalloproteinases (MMPs) 8 and 13 comprise the collagenase subfamily in rats and mice, and only MMP13 has been implicated in degradation of the collagenous matrices during development of bone and cartilage. On the hypothesis that MMP8 is also involved in bone and cartilage development, the present study was designed to investigate gene expression of MMP8 in rat embryonic mandibles and hind limbs. Expression of MMP8 was examined with in situ hybridization and RT-PCR and was compared with that of MMP13. Osteoblastic and chondrocytic cells expressing collagenous matrix molecules were identified using in situ hybridization for collagen Types I and II. The results demonstrated that MMP8 is expressed by osteoblastic progenitors, differentiated osteoblasts, osteocytes, and chondrocytes in the growth plate for the first time. Furthermore, the expression of MMP8 is much broader than that of MMP13, for which expression is confined to differentiated phenotypes of osteoblastic and chondrocytic lineage.