The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Pre-transplant Biomarkers of Immune Dysfunction Improve Risk Assessment of Post-transplant Mortality Compared to Conventional Clinical Risk Scores.

Introduction: There is a critical need to accurately stratify liver transplant (LT) candidates' risk of post-LT mortality prior to LT to optimize patient selection and avoid futility. Here, we compare previously described pre-LT clinical risk scores with the recently developed Liver Immune Frailty Index (LIFI) for prediction of post-LT mortality. LIFI measures immune dysregulation based on pre-LT plasma HCV IgG, MMP3 and Fractalkine. LIFI accurately predicts post-LT mortality, with LIFI-low corresponding to 1.4% 1-year post-LT mortality compared with 58.3% for LIFI-high (C-statistic=0.85). Methods: LIFI was compared to MELD, MELD-Na, MELD 3.0, D-MELD, MELD-GRAIL, MELD-GRAIL-Na, UCLA-FRS, BAR, SOFT, P-SOFT, and LDRI scores on 289 LT recipients based on waitlist data at the time of LT. Survival, hazard of early post-LT death, and discrimination power (C-statistic) were assessed. Results: LIFI showed superior discrimination (highest C-statistic) for post-LT mortality when compared to all other risk scores, irrespective of biologic MELD. On univariate analysis, the LIFI showed a significant correlation with mortality 6-months, as well as 1-, 3-, and 5-years. No other pre-LT scoring system significantly correlated with post-LT mortality. On bivariate adjusted analysis, African American race (p<0.05) and pre-LT cardiovascular disease (p=0.053) were associated with early- and long-term post-LT mortality. Patients who died within 1-yr following LT had a significantly higher incidence of infections, including 30-day and 90-day incidence of any infection, pneumonia, abdominal infections, and UTI (p<0.05). Conclusions: LIFI, which measures pre-LT biomarkers of immune dysfunction, more accurately predicts risk of post-LT futility compared with current clinical predictive models. Pre-LT assessment of immune dysregulation may be critical in predicting mortality after LT and may optimize selection of candidates with lowest risk of futile outcomes.

Altered properties of human adipose-derived mesenchymal stromal cell during continuous in vitro cultivation.

Adipose-derived stromal cells (ASCs) are now recognized as an accessible, abundant, and reliable stem cells for tissue engineering and regenerative medicine. However, ASCs should be expanded long term in order to harvest higher cell number for clinical application. In this study, ASCs isolated from human subcutaneous adipose tissue and senescence after long-term expansion was evaluated. The results showed that following in vitro expansion to the 15th passage, ASCs show changes in morphology (toward the "fried egg" morphology) and decrease in proliferation potential. Nonetheless, ASCs maintained differentiation potential toward osteoblasts, chondrocytes, and adipocytes. The senescent ASCs show impaired migration capacity under the same basal conditions. OXPHOS and glycolysis decreased slightly in culture from passage 5 to passage 15. ASCs also showed increased accumulation of beta-galactosidase in culture. Expression of senescence markers p53, p16, and p21 were also increased accompanied with the increase of passages. Experiment data showed that ASCs biological characteristics depended and changed with age. We recommend the use of early-passage cells, particularly those before passage 5, for efficacious therapeutic application of stem cells.

MicroRNA302-367-PI3K-PTEN-AKT-mTORC1 pathway promotes the development of cardiac hypertrophy through controlling autophagy.

Cardiac hypertrophy at a decompensated state eventually leads to heart failure that mostly contributes to deaths globally. Dysregulated cardiac autophagy is a hallmark of a diseased heart, and a close contact between cardiac autophagy and cardiac hypertrophy is emerging. MicroRNAs (miRNAs) have been recently reported to be prominently implicated in cardiac hypertrophy through regulating cardiac autophagy. However, the role and function of miR302-367 clusters in cardiac autophagy and cardiac hypertrophy remain largely masked. Therefore, to investigate the performance of miR302-367 in cardiac hypertrophy, the specific in vitro hypertrophic model was established in H9c2 cells upon Ang II treatment. Consequently, we discovered a distinct inhibition on autophagy and a remarkable upregulation of miR302-367 expression in hypertrophic H9c2 cells. Besides, loss- and gain-of-function assays demonstrated miR302-367 inhibited autophagy and then aggravated cardiac hypertrophy. Mechanically, PTEN was predicted and confirmed as the shared target of miR302-367. Further, we recognized the apparent inactivation of PI3K/AKT/mTORC1 signaling in the face of miR302-367 suppression in Ang II-induced hypertrophic H9c2 cells. Moreover, co-treatment of PTEN inhibitor re-activated the PI3K/AKT/mTORC1 pathway, therefore counteracting the pro-autophagic and anti-hypertrophic effects of miR302-367 depletion on cardiomyocytes. These findings unveiled the pivotal role of the miR302-367 cluster in regulating cardiac autophagy and therefore modulating cardiac hypertrophy through PTEN/PI3K/AKT/mTORC1 signaling, indicating a promising therapeutic strategy for cardiac hypertrophy and even heart failure. Graphical abstract .

Calibration of the retardation inhomogeneity for the compensator-rotating imaging ellipsometer.

Owing to high accuracy in the whole measurement range, the compensator-rotating method is a main approach for the single-point ellipsometric measurement. The disadvantage of this method is the complexity resulting from the retardation calibration for the rotating compensator. The compensator-rotating imaging ellipsometer, a system combining the optical microscope and the single-point measurement ellipsometer, faces a similar issue. An important problem for the imaging ellipsometer is that the retardation of the compensator manifests inhomogeneity over the view field. Here, we propose a calibration method of the retardation of the compensator for the imaging ellipsometer. The approach was tested experimentally with a homebuilt imaging ellipsometer by generating maps of the ellipsometric parameters $\Delta$Δ and $\psi$ψ of samples of a Si wafer and an opaque Cr thin film.

B Cells Produce the Tissue-Protective Protein RELMα during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphysema.

Emphysema results in destruction of alveolar walls and enlargement of lung airspaces and has been shown to develop during helminth infections through IL-4R-independent mechanisms. We examined whether interleukin 17A (IL-17A) may instead modulate development of emphysematous pathology in mice infected with the helminth parasite Nippostrongylus brasiliensis. We found that transient elevations in IL-17A shortly after helminth infection triggered subsequent emphysema that destroyed alveolar structures. Furthermore, lung B cells, activated through IL-4R signaling, inhibited early onset of emphysematous pathology. IL-10 and other regulatory cytokines typically associated with B regulatory cell function did not play a major role in this response. Instead, at early stages of the response, B cells produced high levels of the tissue-protective protein, Resistin-like molecule α (RELMα), which then downregulated IL-17A expression. These studies show that transient elevations in IL-17A trigger emphysema and reveal a helminth-induced immune regulatory mechanism that controls IL-17A and the severity of emphysema.

MicroRNA-590-5p represses proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

INTRODUCTION: Pediatric asthma has remained a health threat to children in recent years. The abnormal proliferation of airway smooth muscle (ASM) cells contributes to airway remodeling during development of asthma. MicroRNAs (miRNAs) are critical regulators of ASM cell proliferation during airway remodeling. miR-590-5p has been reported to regulate cell proliferation in various cell types. However, it remains unclear whether miR-590-5p regulates ASM cell proliferation. In this study, we aimed to investigate the potential role of miR-590-5p in regulating fetal ASM cell proliferation in vitro stimulated by platelet-derived growth factor (PDGF). MATERIAL AND METHODS: miRNA, mRNA, and protein expression was detected by real-time quantitative polymerase chain reaction and western blot. Cell proliferation was detected by CCK-8 and BrdU assays. The target of miR-590-5p was confirmed by dual-luciferase reporter assay. RESULTS: MiR-590-5p expression was significantly down-regulated in fetal ASM cells stimulated with PDGF (p < 0.05). Overexpression of miR-590-5p inhibited cell proliferation (p < 0.05), whereas the suppression of miR-590-5p promoted cell proliferation of fetal ASM cells stimulated with PDGF (p < 0.05). Signal transducer and activator of transcription 3 (STAT3) was identified as a target gene of miR-590-5p. In addition, miR-590-5p negatively regulated STAT3 expression (p < 0.05). Moreover, miR-590-5p also modulated downstream genes of STAT3 including cyclin D3 and p27 (p < 0.05). The restoration of STAT3 significantly reversed the inhibitory effect of miR-590-5p on fetal ASM cell proliferation. CONCLUSIONS: MiR-590-5p inhibits proliferation of fetal ASM cells by down-regulating STAT3, thereby suggesting a novel therapeutic target for the treatment of pediatric asthma.

MicroRNA-874 inhibits TNF-α-induced remodeling in human fetal airway smooth muscle cells by targeting STAT3.

Pro-inflammatory cytokines-induced airway remodeling was a significant feature of asthma disease. The aim of the present study was to explore the functional significance of miR-874 in tumor necrosis factor (TNF)-α-treated human fetal airway smooth muscle (fASM) cells. Here, we found that TNF-α treatment significantly down-regulated the expression of miR-874 in fASM cells. MiR-874 overexpression markedly inhibited cell viability and migration, suppressed the expression of PCNA and Ki67, reduced the expression of collagen I and collagen III, decreased the expression and activity of matrix metalloproteinase (MMP)-9 and MMP-2, and induced an obvious elevation of tissue inhibitors of metalloproteinases (TIMPs). In addition, the increased production of interleukin (IL)-6, IL-8 and eotaxin induced by TNF-α were significantly inhibited by miR-874 overexpression. Signal transducers and activators of transcription (STAT) 3 was identified as a direct target of miR-874, and STAT3 overexpression partly reversed the protective effects of miR-874 against TNF-α-induced airway remodeling. Overall, these findings demonstrate that miR-874 inhibits TNF-α-induced remodeling in human fASM cells at least in part by targeting STAT3.

miR-205 Inhibits Neuroblastoma Growth by Targeting cAMP-Responsive Element-Binding Protein 1.

Accumulating evidence indicates that microRNA-205 (miR-205) is involved in tumor initiation, development, and metastasis in various cancers. However, its functions in neuroblastoma (NB) remain largely unclear. Here we found that miR-205 was significantly downregulated in human NB tissue samples and cell lines. miR-205 expression was lower in poorly differentiated NB tissues and those of advanced International Neuroblastoma Staging System stage. In addition, restoration of miR-205 in NB cells suppressed proliferation, migration, and invasion and induced cell apoptosis in vitro, as well as impaired tumor growth in vivo. cAMP-responsive element-binding protein 1 (CREB1) was identified as a direct target gene of miR-205. Expression of an miR-205 mimic in NB cells significantly diminished expression of CREB1 and the CREB1 targets BCL-2 and MMP9. CREB1 was also found to be upregulated in human NB tissues, its expression being inversely correlated with miR-205 expression (r = -0.554, p = 0.003). Importantly, CREB1 upregulation partially rescued the inhibitory effects of miR-205 on NB cells. These findings suggest that miR-205 may function as a tumor suppressor in NB by targeting CREB1.

Extraction of polarization properties of the individual components of a layered system by using spectroscopic Mueller matrix analysis.

In semiconductor and optics fields, some devices are constructed with layered systems including two or three individual layers. Measurement of polarization properties of the individual components of these layered systems is often desired. In this paper, we present methods allowing the simultaneous extraction of the polarization parameters of the individual components by analyzing spectroscopic Mueller matrices (measured at two wavelengths). We have studied both retarder-retarder and retarder-polarizer-retarder systems. The validities of the methods were successfully tested using both simulations and real polarization systems.

Correction of large retardation window effect for ellipsometry measurements using quasi-Newton method.

Optical windows attached to the sample chamber for ellipsometry measurements often manifest stress-induced retardation. In reality, the retardation is not always small enough to be simplified with the small angle approximation, varies within the optical beam, and furthermore may change with the evolution of the environmental conditions of the chamber. Large retardations and retardation distributions result in complicated Mueller matrices of the input window-sample-output window system. Consequently, extensive computation is required to obtain the true ellipsometric parameters (Δ,Ψ) of the sample. In this paper, we show that the quasi-Newton method can be used to simultaneously obtain 14 unknown parameters (including ellipsometric parameters) from the Mueller matrix, with a single measurement under conditions of large and inhomogeneous window retardation. A limitation of the method is that it is valid only for isotropic samples. The validity of the method has been tested using a set of samples: a silicon substrate, a TiN-coated silicon wafer, and a thermally grown silicon dioxide film. Measurements were done under the window effect and corrected using the proposed method. The corrected results showed excellent agreement with ellipsometric parameters obtained without window effect.

General window correction method for ellipsometry measurements.

In the ellipsometry measurement, optical windows attached to the sample chamber are often considered as retarders with small retardation. In reality, the retardation is not always small enough to adopt small angle approximation, and furthermore the retardation over optical beam varies point to point. The general window correction method proposed here copes with both the large birefringence and birefringence variance. This method is valid for correction of both isotropic and anisotropic sample measurements. An isotropic reference sample is premeasured by a spectroscopic Mueller matrix ellipsometer to predetermine the effect of windows. The Mueller matrices of samples such as the silicon and low-dielectric constant film in the chamber were measured, and ellipsometric parameters (Δ, Ψ) of these samples were extracted from these matrices, then compared with ex situ measurement results. The ellipsometric parameters of the reflection diffraction grating were also measured under the window effect and corrected by using the proposed method. Excellent agreements of corrected results with ex situ parameters show the significance of this method.

Correction of large birefringent effect of windows for in situ ellipsometry measurements.

To extract true optical properties of samples in a chamber with entrance and exit optical windows, oftentimes the windows were approximated as simple retarders where the retardation was small and premeasured under a given condition. The proposed method allows to cope with large birefringent effect of chamber windows thanks to its capability of extracting ellipsometric parameters (Δ, Ψ) of isotropic samples as well as measuring birefringent parameters (δ, θ) of each window separately and simultaneously. This method is, however, not valid for anisotropic samples. Ex situ results and extracted ellipsometric parameters results from in situ measurements of a silicon substrate and a SiO2 film thermally grown on the silicon substrate exhibited excellent agreement and provided significance of this method.

Does pretreatment of bone marrow mesenchymal stem cells with 5-azacytidine or double intravenous infusion improve their therapeutic potential for dilated cardiomyopathy?

BACKGROUND: This study was designed to investigate whether pretreatment of bone marrow mesenchymal stem cells (BMSCs) with 5-azacytidine (5-aza) or double intravenous infusion could enhance their therapeutic potential for dilated cardiomyopathy (DCM). MATERIAL/METHODS: BMSCs were cultured for 2 weeks in the presence or absence of 5-aza and DCM serum. The cultured BMSCs (Groups 1 and 2), 5-aza-induced BMSCs (Groups 3 and 4), and medium alone (model control) were transplanted into 80 female Wistar rats by intravenous tail vein injection. Double infusion of BMSCs with 1-day time-interval was carried out in Groups 2 and 4. Postmortem histological analysis and evaluation of heart function were performed at 4 weeks post-transplantation. RESULTS: Some transplanted BMSCs engrafted into myocardial tissue and were positive for cardiac marker troponin T. The hearts containing transplanted BMSCs secreted a larger amount of vascular endothelial growth factor. Cardiac function parameters and serum level of brain natriuretic peptide (BNP) did not differ among Groups 1, 3, and the model control. As compared with model control, BMSC transplantation in Groups 2 and 4 significantly decreased the serum level of BNP and improved cardiac contractile function, as evidenced by reduced left ventricular end-diastolic and end-systolic diameter, elevated ejection fraction, and fractional shortening. CONCLUSIONS: BMSC transplantation is a promising strategy for the treatment of DCM. Pretreatment of BMSCs with 5-aza and DCM serum does not enhance their therapeutic efficacy, and the double intravenous BMSC infusion method is superior to single infusion for preserving cardiac contractile function in a rat model of DCM.

Epidemiological investigation of Kawasaki disease in Jilin province of China from 2000 to 2008.

OBJECTIVE: To investigate the epidemiological characteristics of Kawasaki disease in Jilin province of China and explore its clinical features. METHODS: The medical records of children with Kawasaki disease hospitalised in the First Affiliated Hospital of Jilin University and Yanbian University between January, 2000 and December, 2008 were retrospectively analysed. RESULTS: A total of 735 children with Kawasaki disease were enrolled in this study with 483 boys and 252 girls. The ratio of male to female was 1.92:1. The ages of the children at onset varied from 51 days to 12 years with a mean age of 2.8 years. The children under the age of 5 years accounted for 79.5%, but most children were 2-3 years old. Kawasaki disease occurred all the year and more frequently in both the ending of spring and the beginning of summer. Fever was the most common clinical feature and enlarged cervical lymph nodes were the smallest clinical feature. A cardiovascular lesion was found in 41.4% of these children, in whom coronary artery dilatation was the most common (26.97%). A total of 117 (18.2%) of 643 children (87.5%) receiving intravenous immunoglobulin had a non-response to gamma globulin. Of the 117 children, 66 (56.4%) had cardiovascular lesion. Kawasaki disease recurred in 19 children (2.6%). CONCLUSION: The incidence of Kawasaki disease in Jilin province has shown an increasing tendency. The age at onset is slightly higher than that described in other reports. Kawasaki disease is the most common in both the ending of spring and the beginning of summer, and the second incidence peak occurs in autumn.

Polarization properties of scattered light from macrorough surfaces.

We have characterized macrorough surfaces by measuring angle-resolved Stokes parameters of scattering. The analysis of the parameters as a function of a virtual scattering angle shows that polarization properties of the scattering in the plane of incidence display a very strong dependence on the surface roughness. The method and results of this analysis have a significant impact on the application of light scattering to the inspection and process-evaluation industry.