A highly sensitive and specific system for large-scale gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Rapid progress in the field of gene expression-based molecular network integration has generated strong demand on enhancing the sensitivity and data accuracy of experimental systems. To meet the need, a high-throughput gene profiling system of high specificity and sensitivity has been developed.</p> <p>Results</p> <p>By using specially designed primers, the new system amplifies sequences in neighboring exons separated by big introns so that mRNA sequences may be effectively discriminated from other highly related sequences including their genes, unprocessed transcripts, pseudogenes and pseudogene transcripts. Probes used for microarray detection consist of sequences in the two neighboring exons amplified by the primers. In conjunction with a newly developed high-throughput multiplex amplification system and highly simplified experimental procedures, the system can be used to analyze >1,000 mRNA species in a single assay. It may also be used for gene expression profiling of very few (<it>n </it>= 100) or single cells. Highly reproducible results were obtained from duplicate samples with the same number of cells, and from those with a small number (100) and a large number (10,000) of cells. The specificity of the system was demonstrated by comparing results from a breast cancer cell line, MCF-7, and an ovarian cancer cell line, NCI/ADR-RES, and by using genomic DNA as starting material.</p> <p>Conclusion</p> <p>Our approach may greatly facilitate the analysis of combinatorial expression of known genes in many important applications, especially when the amount of RNA is limited.</p

Lipopolysaccharide (LPS) potentiates hydrogen peroxide toxicity in T98G astrocytoma cells by suppression of anti-oxidative and growth factor gene expression

<p>Abstract</p> <p>Background</p> <p>Lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria with proved role in pathogenesis of sepsis. Brain injury was observed with both patients dead from sepsis and animal septic models. However, <it>in vitro </it>administration of LPS has not shown obvious cell damage to astrocytes and other relative cell lines while it does cause endothelial cell death <it>in vitro</it>. These observations make it difficult to understand the role of LPS in brain parenchymal injury.</p> <p>Results</p> <p>To test the hypothesis that LPS may cause biological changes in astrocytes and make the cells to become vulnerable to reactive oxygen species, a recently developed highly sensitive and highly specific system for large-scale gene expression profiling was used to examine the gene expression profile of a group of 1,135 selected genes in a cell line, T98G, a derivative of human glioblastoma of astrocytic origin. By pre-treating T98G cells with different dose of LPS, it was found that LPS treatment caused a broad alteration in gene expression profile, but did not cause obvious cell death. However, after short exposure to H₂O₂, cell death was dramatically increased in the LPS pretreated samples. Interestingly, cell death was highly correlated with down-regulated expression of antioxidant genes such as cytochrome b561, glutathione s-transferase a4 and protein kinase C-epsilon. On the other hand, expression of genes encoding growth factors was significantly suppressed. These changes indicate that LPS treatment may suppress the anti-oxidative machinery, decrease the viability of the T98G cells and make the cells more sensitive to H₂O₂stress.</p> <p>Conclusion</p> <p>These results provide very meaningful clue for further exploring and understanding the mechanism underlying astrocyte injury in sepsis <it>in vivo</it>, and insight for why LPS could cause astrocyte injury <it>in vivo</it>, but not <it>in vitro</it>. It will also shed light on the therapeutic strategy of sepsis.</p

A Large Portal Vein: A Rare Finding of Recent Portal Vein Thrombosis

Acute portal vein thrombosis (PVT) is rarely encountered by clinicians. The most common manifestation of acute PVT is sudden onset of abdominal pain. A computed tomography scan without contrast often shows a high-density material in the portal vein. After injection of contrast agents, absence of luminal enhancement and enlargement of the obstructed portal vein are shown. In this case report, we demonstrated a rare computed tomography finding in which the diameter of the main portal vein was enormously distended to 3-fold that of the aorta in a patient with recent PVT. Despite thrombolysis and anticoagulation were immediately given, portal venous recanalization was not achieved in the patient. After 5 years, variceal bleeding and ascites occurred and liver function had persistently deteriorated. Finally, he died of progressive liver failure. Considering this case, we suggest that an early decision for invasive interventional treatment might be necessary to both increase the rate of portal venous recanalization and improve prognosis, as anticoagulation and thrombolysis therapy failed to recanalize recent PVT

Trajectories of Self-Rated Health of Chinese Elders: A Piecewise Growth Model Analysis

This study used piecewise growth modeling to describe the developmental trajectories of self-rated health (SRH) in the elderly and longitudinal associations with activities of daily living (ADL), educational level, economic status, age, and gender. Data were drawn from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), collected over 12 years (from 2002 to 2014) at five waves. A total of 16,064 Chinese elders (57.4% females) were analyzed. Results showed two phases of development for SRH; specifically, the decreasing trend of SRH was from slow (in the first phase, waves 1 to 3) to fast (in the second phase, waves 3 to 5). Descriptives showed that the turning point age was at the age of 83.69 (range = 68 to 116, median age = 82 years old). ADL were positively associated with SRH within each time point (wave of data). Female elders had a higher initial state (i.e., worse) of SRH than did male elders, and poorer economic status was associated with worse initial status of SRH

Effects of Salmonella enterica serovar Enteritidis infection on egg production and the immune response of the laying duck Anas platyrhynchos

Persistent colonization of the avian reproductive tract by Salmonella enterica serovar Enteritidis (SE) negatively affects egg production and contaminates the egg. The immune function of the ovary and oviduct is essential for protection from infection and for the production of wholesome eggs. However, the immune response of laying ducks during SE infection is not well-understood. In this study, ducks (Anas platyrhynchos) were infected with SE and were systematically monitored for fecal shedding during a 13-week period. We also assessed bacterial distribution in the reproductive tract and classified infected ducks as resistant or susceptible based on the presence of tissue lesions and on SE isolation from fecal samples. We found that infected animals had persistent, but intermittent, bacterial shedding that resulted in the induction of carrier ducks. Laying rate and egg quality were also decreased after SE infection (P < 0.05). SE readily colonized the stroma, small follicle, isthmus, and vagina in the reproductive tracts of susceptible ducks. Immunoglobulin (IgA, IgG, IgM) levels were higher in susceptible ducks compared with resistant birds (P < 0.05); T-lymphocyte subpopulations (CD3+, CD4+, CD8+) displayed the opposite trend. qRT-PCR analysis was used to examine expression profiles of immune response genes in the reproductive tract of infected ducks. The analysis revealed that immune genes, including toll-like receptors (TLR2, TLR4-5, TLR15, TLR21), NOD-like receptors (NOD1, NLRX1, NLRP12), avian β-defensins (AvβD4-5, AvβD7, AvβD12), cytokines (IL-6, IL-1β, IFN-γ), and MyD88 were markedly upregulated in the reproductive tracts of SE-infected ducks (all P < 0.05); TLR3, TLR7, NLRC3, NLRC5, and TNF-α were significantly downregulated. These results revealed that SE infection promoted lower egg production and quality, and altered the expression of TLRs, NLRs, AvβDs, and cytokine family genes. These findings provide a basis for further investigation of the physiological and immune mechanisms of SE infection in laying ducks