Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

Education for Children With ASD in South Korea: A Case Study.

In South Korea, there is currently a massive gap between the demand and the supply of quality applied behavior analysis (ABA) services for children with autism spectrum disorder (ASD) and their families. However, the literature on the implementation and effectiveness of ABA intervention mainly comes from Western countries, and the voices of Asian countries are scarcely heard. The present article reports data collected from the KAVBA Center in Seoul, South Korea, as a direct replication of the CABAS educational model. Eleven 3- to 4-year-old children with ASD were the participants in the study and attended the center for 1 year. Our pre- and postintervention data show that the CABAS model provided an effective and cost-efficient service for children with ASD in South Korea.

A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice.

tRNA-derived small RNAs (tsRNAs) have been implicated in many cellular processes, yet the detailed mechanisms are not well defined. We previously found that the 3' end of Leu-CAG tRNA-derived small RNA (LeuCAG3'tsRNA) regulates ribosome biogenesis in humans by maintaining ribosomal protein S28 (RPS28) levels. The tsRNA binds to coding (CDS) and non-coding 3' UTR sequence in the RPS28 mRNA, altering its secondary structure and enhancing its translation. Here we report that the functional 3' UTR target site is present in primates while the CDS target site is present in many vertebrates. We establish that this tsRNA also regulates mouse Rps28 translation by interacting with the CDS target site. We further establish that the change in mRNA translation occurred at a post-initiation step in both species. Overall, our results suggest that LeuCAG3'tsRNA might maintain ribosome biogenesis through a conserved gene regulatory mechanism in vertebrates.

A transfer-RNA-derived small RNA regulates ribosome biogenesis.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Comparison study of reconstruction algorithms for prototype digital breast tomosynthesis using various breast phantoms.

Digital breast tomosynthesis (DBT) is a recently developed system for three-dimensional imaging that offers the potential to reduce the false positives of mammography by preventing tissue overlap. Many qualitative evaluations of digital breast tomosynthesis were previously performed by using a phantom with an unrealistic model and with heterogeneous background and noise, which is not representative of real breasts. The purpose of the present work was to compare reconstruction algorithms for DBT by using various breast phantoms; validation was also performed by using patient images. DBT was performed by using a prototype unit that was optimized for very low exposures and rapid readout. Three algorithms were compared: a back-projection (BP) algorithm, a filtered BP (FBP) algorithm, and an iterative expectation maximization (EM) algorithm. To compare the algorithms, three types of breast phantoms (homogeneous background phantom, heterogeneous background phantom, and anthropomorphic breast phantom) were evaluated, and clinical images were also reconstructed by using the different reconstruction algorithms. The in-plane image quality was evaluated based on the line profile and the contrast-to-noise ratio (CNR), and out-of-plane artifacts were evaluated by means of the artifact spread function (ASF). Parenchymal texture features of contrast and homogeneity were computed based on reconstructed images of an anthropomorphic breast phantom. The clinical images were studied to validate the effect of reconstruction algorithms. The results showed that the CNRs of masses reconstructed by using the EM algorithm were slightly higher than those obtained by using the BP algorithm, whereas the FBP algorithm yielded much lower CNR due to its high fluctuations of background noise. The FBP algorithm provides the best conspicuity for larger calcifications by enhancing their contrast and sharpness more than the other algorithms; however, in the case of small-size and low-contrast microcalcifications, the FBP reduced detectability due to its increased noise. The EM algorithm yielded high conspicuity for both microcalcifications and masses and yielded better ASFs in terms of the full width at half maximum. The higher contrast and lower homogeneity in terms of texture analysis were shown in FBP algorithm than in other algorithms. The patient images using the EM algorithm resulted in high visibility of low-contrast mass with clear border. In this study, we compared three reconstruction algorithms by using various kinds of breast phantoms and patient cases. Future work using these algorithms and considering the type of the breast and the acquisition techniques used (e.g., angular range, dose distribution) should include the use of actual patients or patient-like phantoms to increase the potential for practical applications.

Effective DQE (eDQE) and dose to optimize radiographic technical parameters: a survey of pediatric chest X-ray examinations in Korea.

OBJECTIVE: The purpose of this study was to investigate the effect of various technical parameters for dose optimization in pediatric chest radiological examinations by evaluating effective dose and effective detective quantum efficiency (eDQE). MATERIALS AND METHODS: For tube voltages ranging from 40 to 90 kV in 10 kV increments at the focus-to-detector distance (FDD) of 100, 110, 120, 150, 180 cm, the eDQE was evaluated at same effective dose. RESULTS: The eDQE was considerably higher without the use of the grid on equivalent effective dose. This indicates that the reduction of scatter radiation did not compensate for the loss of absorbed effective photons in the grid. The eDQE increased with increasing FDD because of the greater effective modulation transfer function (eMTF) with lower focal spot blurring. However, most of the major hospitals in Korea employed a short FDD of 100 cm with the grid. The entrance surface air kerma values for the hospitals of this survey exceeded the Korean reference level of 100 µGy. CONCLUSIONS: The different reference levels might be appropriate for the same examination conducted on children of different ages. Also, it is necessary to refine the technical parameters to perform pediatric chest examinations.

Optimization of configuration parameters in a newly developed digital breast tomosynthesis system.

The purpose of the present work was to investigate the effects of variable projection-view (PV) and angular dose (AD) distributions on the reconstructed image quality for improving microcalcification detection. The PV densities at central and peripheral sites were varied through the distribution of 21 PVs acquired over ± 25° angular range. To vary the AD distribution, 7 PVs in the central region were targeted with two, four and six times the peripheral dose, and the number of central PVs receiving four times the peripheral dose was increased from 3 to 11. The contrast-to-noise ratio (CNR) for in-focus plane quality and the full width at half maximum (FWHM) of artifact spread function (ASF) for resolution in the z-direction were used. Although the ASF improved with increasing PV densities at two peripheral sites, the CNRs were inferior to those obtained with other subsets. With increasing PV density in the central area, the vertical resolution decreased but the CNR increased. Although increasing the central PV or AD concentrations improved image quality, excessive central densities reduced image quality by increasing noise in peripheral views.

Optimization of the key imaging parameters for detection of microcalcifications in a newly developed digital breast tomosynthesis system.

PURPOSE: The purpose of this study was to investigate the effect of different acquisition parameters and to characterize their relationships in order to improve the detection of microcalcifications using digital breast tomosynthesis (DBT). MATERIALS AND METHODS: DBT imaging parameters were optimized using 32 different acquisition sets with 6 angular ranges (± 5°, ± 10°, ± 13°, ± 17°, ± 21°, and ± 25°) and 8 projection views (PVs) (5, 11, 15, 21, 25, 31, 41, and 51 projections). To investigate the effects of variable angular dose distribution, the acquisition sets were evaluated with delivering more dose toward the central views. RESULTS: Our results show that a wide angular range improved the reconstructed image quality in the z-direction. If a large number of projections are acquired, then electronic noise may dominate the contrast-to-noise ratio (CNR) due to reduced radiation dose per projection. With delivering more dose toward the central views, it was found that the vertical resolution was reduced with increasing dose in the central PVs. On the other hand, the CNR clearly increased with increasing concentration of dose distribution in central views. CONCLUSIONS: Although it was found that increasing angular range improved the vertical resolution, it was also found that the image quality of microcalcifications in the in-focus plane did not improve by increasing the noise due to greater effective breast thickness. Angular dose distributions, with more dose delivered to the central views, generally yielded a higher quality factor than uniform dose distributions.

Effects of breast thickness and lesion location on resolution in digital magnification mammography.

This study aimed to examine the resolution effects of breast thickness and lesion location in magnification mammography by evaluating generalized modulation transfer function (GMTF) including the effect of focal spot, effective pixel size, and the scatter. Polymethyl methacrylate (PMMA) thicknesses ranging from 10 to 40 mm were placed on a standard supporting platform that was positioned to achieve magnification factors ranging from 1.2 to 2.0. As the magnification increased, the focal spot MTF degraded, while the detector MTF improved. The GMTF depended on the trade-off between the focal spot size and effective pixel size. Breast thickness and lesion location had little effect on the resolution at high frequencies. The resolution of small focal spot did improve slightly with increasing PMMA thickness for magnification factors less than 1.8. In contrast, system resolution decreased with increasing PMMA thickness for magnification factors greater than 1.8 since focal spot blur begins to dominate spatial resolution. In particular, breast thickness had a large effect on the resolution at lower frequencies. A low-frequency drop effect increased with increasing PMMA thickness because of the increase in scatter fraction. Hence, the effect of compressed breast thickness should be considered for the standard magnification factor of 1.8 that is most commonly used in clinical practice. Our results should provide insights for determining optimum magnification in clinical application of digital mammography, and our approaches can be extended to a wide diversity of radiological imaging systems.

Protective role of reactive oxygen species in endotoxin-induced lung inflammation through modulation of IL-10 expression.

Reactive oxygen species (ROS) generated by NADPH oxidase are generally known to be proinflammatory, and it seems to be counterintuitive that ROS play a critical role in regulating the resolution of the inflammatory response. However, we observed that deficiency of the p47(phox) component of NADPH oxidase in macrophages was associated with a paradoxical accentuation of inflammation in a whole animal model of noninfectious sepsis induced by endotoxin. We have confirmed this observation by interrogating four separate in vivo models that use complementary methodology including the use of p47(phox-/-) mice, p47(phox-/-) bone marrow chimera mice, adoptive transfer of macrophages from p47(phox-/-) mice, and an isolated perfused lung edema model that all point to a relationship between excessive acute inflammation and p47(phox) deficiency in macrophages. Mechanistic data indicate that ROS deficiency in both cells and mice results in decreased production of IL-10 in response to treatment with LPS, at least in part, through attenuation of the Akt-GSK3-ß signal pathway and that it can be reversed by the administration of rIL-10. Our data support the innovative concept that generation of ROS is essential for counterregulation of acute lung inflammation.

Functional PU.1 in macrophages has a pivotal role in NF-κB activation and neutrophilic lung inflammation during endotoxemia.

Although the role of ETS family transcriptional factor PU.1 is well established in macrophage maturation, its role in mature macrophages with reference to sepsis- related animal model has not been elucidated. Here, we report the in vivo function of PU.1 in mediating mature macrophage inflammatory phenotype by using bone marrow chimera mice with conditional PU.1 knockout. We observed that the expression of monocyte/macrophage-specific markers CD 11b, F4/80 in fetal liver cells, and bone marrow-derived macrophages were dependent on functional PU.1. Systemic inflammation as measured in terms of NF-κB reporter activity in lung, liver, and spleen tissues was significantly decreased in PU.1-deficient chimera mice compared with wild-type chimeras on lipopolysaccharide (LPS) challenge. Unlike wild-type chimera mice, LPS challenge in PU.1-deficient chimera mice resulted in decreased lung neu-trophilic inflammation and myeloperoxidase activity. Similarly, we found attenuated inflammatory gene expression (cyclooxygenase-2, inducible nitric-oxide synthase, and TLR4) and inflammatory cytokine secretion (IL-6, MCP-1, IL-1ß, TNF-α, and neutrophilic chemokine keratinocyte-derived chemokine) in PU.1-deficient mice. Most importantly, this attenuated lung and systemic inflammatory phenotype was associated with survival benefit in LPS-challenged heterozygotic PU.1-deficient mice, establishing a novel protective mechanistic role for the lineage-specific transcription factor PU.1.

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells.

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.

Replication of Theiler's virus requires NF-kappa B-activation: higher viral replication and spreading in astrocytes from susceptible mice.

To investigate viral replication and cell-cell spreading in astrocytes, recombinant Theiler's murine encephalomyelitis virus (TMEV) expressing green fluorescent protein (GFP) during the replication was generated. GFP and TMEV proteins were processed correctly in infected cells and production of viral proteins could be tracked by fluorescent microscopy. Viral replication of both wild-type TMEV and GFP-TMEV was dependent on the activation of NF-kappaB and partially MAP kinase, based on chemical inhibition studies. Viral replication was significantly reduced in primary astrocytes from NF-kappaB1 (p105)-deficient mice compared with that from wild-type control mice, whereas cytokine production was enhanced. These results suggest an association of canonical NF-kappaB subunits in viral replication, but not cytokine production. Viral replication was also suppressed in both IKKalpha and IKKbeta-deficient mouse embryonic fibroblasts (MEFs), compared with that in wild-type MEF. However, the inhibition was significantly greater in IKKbeta-deficient MEF, suggesting that IKKbeta plays a stronger role in supporting viral replication. Interestingly, viral replication and spreading in primary astrocytes from susceptible SJL/J mice were several-fold higher than those in astrocytes from resistant C57BL/6 mice, suggesting that higher viral replication levels in astrocytes may also contribute to the viral persistence in the central nervous system (CNS) of susceptible SJL/J mice. A relatively higher level of activated NF-kappaB was found in the nuclei of virus-infected SJL astrocytes compared with C57BL/6 astrocytes suggest that the NF-kappaB activation level affects on viral replication.