Early Blood Biomarkers Distinguish Inflammation from Neonatal Hypoxic-Ischemia Encephalopathy

Neurochemical Research - Neonatal hypoxic–ischemic encephalopathy is the most common cause of neurological disability in infancy. Superimposed inflammation may further worsen neurological...     

Monocyte Trafficking,Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue – Evaluations on Monocyte-Based Delivery System for the Central Nervous System

The ability of monocytes and monocyte-derived macrophages (MDM) to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB). This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV) cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP) gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported the notion to use monocytes as a non-invasive cell-based delivery system for the brain.     

A New Simple Method for Improving QTL Mapping Under Selective Genotyping

The selective genotyping approach, where only individuals from the high and low extremes of the trait distribution are selected for genotyping and the remaining individuals are not genotyped, has been known as a cost-saving strategy to reduce genotyping work and can still maintain nearly equivalent efficiency to complete genotyping in QTL mapping. We propose a novel and simple statistical method based on the normal mixture model for selective genotyping when both genotyped and ungenotyped individuals are fitted in the model for QTL analysis. Compared to the existing methods, the main feature of our model is that we first provide a simple way for obtaining the distribution of QTL genotypes for the ungenotyped individuals and then use it, rather than the population distribution of QTL genotypes as in the existing methods, to fit the ungenotyped individuals in model construction. Another feature is that the proposed method is developed on the basis of a multiple-QTL model and has a simple estimation procedure similar to that for complete genotyping. As a result, the proposed method has the ability to provide better QTL resolution, analyze QTL epistasis, and tackle multiple QTL problem under selective genotyping. In addition, a truncated normal mixture model based on a multiple-QTL model is developed when only the genotyped individuals are considered in the analysis, so that the two different types of models can be compared and investigated in selective genotyping. The issue in determining threshold values for selective genotyping in QTL mapping is also discussed. Simulation studies are performed to evaluate the proposed methods, compare the different models, and study the QTL mapping properties in selective genotyping. The results show that the proposed method can provide greater QTL detection power and facilitate QTL mapping for selective genotyping. Also, selective genotyping using larger genotyping proportions may provide roughly equivalent power to complete genotyping and that using smaller genotyping proportions has difficulties doing so. The R code of our proposed method is available on http://www.stat.sinica.edu.tw/chkao/.     

Effects of postexercise supplementation of chicken essence on the elimination of exercise-induced plasma lactate and ammonia

We investigated the effects of chicken essence (CE) supplementation on exercise-induced changes of lactate and ammonia during recovery. In this randomized, double blind, crossover study, twelve healthy subjects performed a single bout of exercise to exhaustion, and then consumed either a placebo or CE within 5-min of the exercise cessation. Blood samples were collected before exercise, at exhaustion (0 minute), and 20, 40, 60, and 120 minutes, respectively during the recovery period. There were no differences in plasma glucose, creatine kinase, or heart rate responses between treatments. The exercise exhaustion significantly increased the levels of lactate and ammonia, and both measured values gradually declined during the recovery period. Ammonia levels at 40, 60, and 120 min. of the recovery period were observed lower significantly in the CE group, as compared to those in the placebo group. Additionally, lactate concentrations at 60 and 120 min were lower in the CE group, as compared to those in the placebo group. In conclusion, the main finding of this study was that CE supplementation after exercise reduces plasma lactate and ammonia levels. The results indicated that CE supplementation after an exhaustive exercise could enhance physiological recovery in humans.     

Cryovial with partial membrane sealing can prevent liquid nitrogen penetration in submerged storage

Cryopreservation is one of the fundamental techniques in life science. To preserve the viability of cells and tissues, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the non-sterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. To prevent these drawbacks while retaining the benefit of constant ultra-low temperature in submerged liquid nitrogen, we used a heat-sealable membrane to cover the upper portion of vials. After heat-sealing, the vials were completely free of liquid nitrogen penetration in the submerging test. Moreover, the sealing process did not affect the cell viability. This modified protocol provides an easy and efficient tool to ensure the integrity of biospecimens in long-term storage without interfering with existing cryobox storage systems.     

Pigment Epithelium-Derived Factor 34-mer Peptide Prevents Liver Fibrosis and Hepatic Stellate Cell Activation through Down-Regulation of the PDGF Receptor

Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl₄)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.     

The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2–20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two “negative” regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.     

A pathway for tumor necrosis factor-alpha-induced Bcl10 nuclear translocation. Bcl10 is up-regulated by NF-kappaB and phosphorylated by Akt1 and then complexes with Bcl3 to enter the nucleus

Bcl10 overexpression and nuclear translocation were originally identified in mucosa-associated lymphoid tissue lymphoma with t(1;14)(p32;q32) chromosome translocation. DNA amplification of Bcl10 was also found in other solid tumors. We have recently shown that nuclear translocation of Bcl10 is a specific molecular determinant of Helicobacter pylori-independent mucosa-associated lymphoid tissue lymphoma (Kuo, S.-H., Chen, L. T., Yeh, K.-H., Wu, M. S., Hsu, H. C., Yeh, P. Y., Mao, T. L., Chen, C. L., Doong, S. L., Lin, J. T., and Cheng, A.-L. (2004) J. Clin. Oncol. 22, 3491-3497). However, the molecular mechanism of Bcl10 nuclear translocation remains unknown. In this study, we observed that tumor necrosis factor-alpha (TNFalpha) up-regulates the expression of Bcl10 and induces a fraction of Bcl10 nuclear translocation in human breast carcinoma MCF7 cells. Chromatin immunoprecipitation assays and electrophoretic mobility shift assays indicated that an NF-kappaB-binding site resides in the Bcl10 5 '-untranslated region. This study also demonstrates that Akt1, activated by TNFalpha, phosphorylates Bcl10 at Ser218 and Ser231 and that phosphorylated Bcl10 subsequently complexes with Bcl3 to enter the nucleus. Either inhibition of Akt1 or depletion of Bcl3 blocks Bcl10 nuclear translocation. In summary, these findings characterize a molecular linkage that directs Bcl10 nuclear translocation in response to TNFalpha treatment.     

Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133⁺) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133⁻ cells. To evaluate the role of SirT1 in GBM-CD133⁺, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133⁺. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133⁺ to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133⁺. Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133⁺ mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.