Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been ...implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.
Objective
The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in ...patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.
Hypothesis
We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.
Methods
We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.
Results
Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.
Conclusions
Together, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.
Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was ...over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes.
To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity.
One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior.
Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be especially sensitive to changes in SERT function. Finally, these results provide a platform for the identification of genes that may modulate the risk of ASD in humans.
The disappearance of reactor
ν
¯
e
observed by the Daya Bay experiment is examined in the framework of a model in which the neutrino is described by a wave packet with a relative intrinsic momentum ...dispersion
σ
rel
. Three pairs of nuclear reactors and eight antineutrino detectors, each with good energy resolution, distributed among three experimental halls, supply a high-statistics sample of
ν
¯
e
acquired at nine different baselines. This provides a unique platform to test the effects which arise from the wave packet treatment of neutrino oscillation. The modified survival probability formula was used to fit Daya Bay data, providing the first experimental limits:
2.38
×
10
-
17
<
σ
rel
<
0.23
. Treating the dimensions of the reactor cores and detectors as constraints, the limits are improved:
10
-
14
≲
σ
rel
<
0.23
, and an upper limit of
σ
rel
<
0.20
(which corresponds to
σ
x
≳
10
-
11
cm
) is obtained. All limits correspond to a 95% C.L. Furthermore, the effect due to the wave packet nature of neutrino oscillation is found to be insignificant for reactor antineutrinos detected by the Daya Bay experiment thus ensuring an unbiased measurement of the oscillation parameters
sin
2
2
θ
13
and
Δ
m
32
2
within the plane wave model.
Unusually large CAG repeat expansions (>60) in exon one of Huntingtin (HTT) are invariably associated with a juvenile-onset form of Huntington's disease (HD), characterized by a more extensive and ...rapidly progressing neuropathology than the more prevalent adult-onset form. However, existing mouse models of HD that express the full-length Htt gene with CAG repeat lengths associated with juvenile HD (ranging between ~75 to ~150 repeats in published models) exhibit selective neurodegenerative phenotypes more consistent with adult-onset HD. Objective: To determine if a very large CAG repeat (>200) in full-length Htt elicits neurodegenerative phenotypes consistent with juvenile HD.
Using a …bacterial artificial chromosome (BAC) system, we generated mice expressing full-length mouse Htt with ~225 CAG repeats under control of the mouse Htt promoter. Mice were characterized using behavioral, neuropathological, biochemical and brain imaging methods.
BAC-225Q mice exhibit phenotypes consistent with a subset of features seen in juvenile-onset HD: very early motor behavior abnormalities, reduced body weight, widespread and progressive increase in Htt aggregates, gliosis, and neurodegeneration. Early striatal pathology was observed, including reactive gliosis and loss of dopamine receptors, prior to detectable volume loss. HD-related blood markers of impaired energy metabolism and systemic inflammation were also increased. Aside from an age-dependent progression of diffuse nuclear aggregates at 6 months of age to abundant neuropil aggregates at 12 months of age, other pathological and motor phenotypes showed little to no progression.
The HD phenotypes present in animals 3 to 12 months of age make the BAC-225Q mice a unique and stable model of full-length mutant Htt associated phenotypes, including body weight loss, behavioral impairment and HD-like neurodegenerative phenotypes characteristic of juvenile-onset HD and/or late-stage adult-onset HD.
The influence of 6 different cooling rates (1.4, 5, 10, 15, 20, 160 K/min) and 5 different compositions of the suspensions to be frozen (% DMSO/% HES: 10/0, 7.5/2.5, 5/6, 2.5/7.5, 0/10) were ...investigated in 30 aliquots from each of 12 peripheral blood progenitor cells (PBSC) products which had been obtained by leukapheresis from 11 patients with hematological malignancies and solid tumors and 1 healthy donor treated with 5-24 micrograms/kg body weight/day granulocyte colony stimulating factor (G-CSF) over 5 days. The MNC concentration in the products obtained amounted to 4.51 +/- 1.55 x 10(7)/ml (mean +/- SEM), range: 2.10-7.65 x 10(7)/ml). For freezing, cooling rates were generated by means of a liquid nitrogen(LN2)-operated, computer-controlled freezer, a mechanical -80 degrees C freezer, in the vapor phase over LN2, and by submerging into LN2. The statistical analysis of the results clearly indicates that optimum results compared with the prefreeze values for numerical recovery (80.6 +/- 20.1%, Coulter counter), viability (membrane integrity by Trypan blue exclusion 91.6 +/- 4.1%), and colony-forming potential (56.2 +/- 45.8% erythroid burst-forming units BFU-E, 63.4 +/- 72.8% myeloid colonies CFU-GM) were achieved at a cooling rate of 1.4 K/min with 10% DMSO being present. The values obtained at a cooling rate of 5 K/min (-80 degrees C mechanical refrigerator) in the presence of 5% DMSO and 6% HES did not differ significantly (i.e., membrane integrity 91.8 +/- 3.9%, BFU-E 53.0 +/- 37.4%, CFU-GM 47.8 +/- 58.2%). At cooling rates above 5 K/min and DMSO concentrations lower than 5% (in spite of higher HES concentrations) there was a significant drop of CFU recovery (CFU-GM plus BFU-E) to almost 0%. In parallel, numerical recovery and viability dropped as well, but less pronounced, indicating that both methods are unsuitable for the prediction of CFU recovery when different freezing protocols are applied. We need at least 5% DMSO (in the presence of 6% HES) in spite of the undesirable histamine-releasing effect of this compound. The cooling rate is not critical in the range from 1 to 5 K/min and can easily be achieved by -80 degrees C freezers.
Birkhoff Interpolation Lorentz, G. G.; Jetter, K.; Riemenschneider, S. D.
12/1984, Letnik:
v.Series Number 19
eBook
This reference book provides the main definitions, theorems and techniques in the theory of Birkhoff interpolation by polynomials. The book begins with an article by G. G. Lorentz that discusses some ...of the important developments in approximation and interpolation in the last twenty years. It presents all the basic material known at the present time in a unified manner. Topics discussed include; applications of Birkhoff interpolation to approximation theory, quadrature formulas and Chebyshev systems; lacunary interpolation at special knots and an introduction to the theory of Birkhoff interpolation by splines.
PDF
