Analysis of multiple sequence alignments can generate important, testable hypotheses about the phylogenetic history and cellular function of genomic sequences. We describe the MultiPipMaker server, which aligns multiple, long genomic DNA sequences quickly and with good sensitivity (available at http://bio.cse.psu.edu/ since May 2001). Alignments are computed between a contiguous reference sequence and one or more secondary sequences, which can be finished or draft sequence. The outputs include a stacked set of percent identity plots, called a MultiPip, comparing the reference sequence with subsequent sequences, and a nucleotide-level multiple alignment. New tools are provided to search MultiPipMaker output for conserved matches to a user-specified pattern and for conserved matches to position weight matrices that describe transcription factor binding sites (singly and in clusters). We illustrate the use of MultiPipMaker to identify candidate regulatory regions in WNT2 and then demonstrate by transfection assays that they are functional. Analysis of the alignments also confirms the phylogenetic inference that horses are more closely related to cats than to cows.

BackgroundIt has been suggested that p300 participates in the regulation of a wide range of cell biological processes and mutation of p300 has been identified in certain types of human cancers. However, the expression dynamics of p300 in hepatocellular carcinoma (HCC) and its clinical/prognostic significance are unclear.MethodsIn this study, the methods of reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry (IHC) were utilized to investigate protein/mRNA expression of p300 in HCCs. Receiver operating characteristic (ROC) curve analysis, spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data.ResultsUp-regulated expression of p300 mRNA and protein was observed in the majority of HCCs by RT-PCR and Western blotting, when compared with their adjacent non-malignant liver tissues. According to the ROC curves, the cutoff score for p300 high expression was defined when more than 60% of the tumor cells were positively stained. High expression of p300 was examined in 60/123 (48.8%) of HCCs and in 8/123 (6.5%) of adjacent non-malignant liver tissues. High expression of p300 was correlated with higher AFP level, larger tumor size, multiplicity, poorer differentiation and later stage (P < 0.05). In univariate survival analysis, a significant association between overexpression of p300 and shortened patients' survival was found (P = 0.001). In different subsets of HCC patients, p300 expression was also a prognostic indicator in patients with stage II (P = 0.007) and stage III (P = 0.011). Importantly, p300 expression was evaluated as an independent prognostic factor in multivariate analysis (P = 0.021). Consequently, a new clinicopathologic prognostic model with three poor prognostic factors (p300 expression, AFP level and vascular invasion) was constructed. The model could significantly stratify risk (low, intermediate and high) for overall survival (P < 0.0001).ConclusionsOur findings provide a basis for the concept that high expression of p300 in HCC may be important in the acquisition of an aggressive phenotype, suggesting that p300 overexpression, as examined by IHC, is an independent biomarker for poor prognosis of patients with HCC. The combined clinicopathologic prognostic model may become a useful tool for identifying HCC patients with different clinical outcomes.

ABSTRACT:The metabolism, excretion, and pharmacokinetics of 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (INCB018424), a potent, selective inhibitor of Janus tyrosine kinase1/2 and the first investigational drug of its class in phase III studies for the treatment of myelofibrosis, were investigated in healthy human subjects given a single oral 25-mg dose of [ 14 C]INCB018424 as an oral solution. INCB018424 and total radioactivity were absorbed rapidly (mean time to reach the maximal drug concentration <1 h), declining in a monophasic or biphasic fashion (mean t 1/2 of 2.32 and 5.81 h, respectively). Recovery of administered radioactivity was fairly rapid (>70% within 24 h postdose) with 74 and 22% recovered in urine and feces, respectively. Parent compound was the predominant entity in the circulation, representing 58 to 74% of the total radioactivity up to 6 h postdose, indicating that the overall circulating metabolite burden was low (<50% of parent). Two metabolite peaks in plasma (M18 and a peak containing M16/M27, both hydroxylations on the cyclopentyl moiety) were identified as major (30 and 14% of parent based on area under the curve from 0 to 24 h). The exposures of other circulating INCB018424-related peaks were <10% of parent, consisting of mono-and dihydroxylated metabolites. The profiles in urine and feces consisted of hydroxyl and oxo metabolites and subsequent glucuronide conjugates with parent drug accounting for <1% of the excreted dose, strongly suggesting that after an oral dose, INCB018424 was >95% absorbed. In healthy subjects administered daily oral doses of unlabeled INCB018424, there were minimal differences in parent and metabolite concentrations between day 1 and day 10, indicating a lack of accumulation of parent or metabolites between single and multiple dosing.

cAberrant mucin secretion and accumulation in the airway lumen are clinical hallmarks associated with various lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Mycoplasma pneumoniae, long appreciated as one of the triggers of acute exacerbations of chronic pulmonary diseases, has recently been reported to promote excessive mucus secretion. However, the mechanism of mucin overproduction induced by M. pneumoniae remains unclear. This study aimed to determine the mechanism by which M. pneumoniae induces mucus hypersecretion by using M. pneumoniae infection of mouse lungs, human primary bronchial epithelial (NHBE) cells cultured at the air-liquid interface, and the conventionally cultured airway epithelial NCI-H292 cell line. We demonstrated that M. pneumoniae induced the expression of mucins MUC5AC and MUC5B by activating the STAT6-STAT3 and epidermal growth factor receptor (EGFR) signal pathways, which in turn downregulated FOXA2, a transcriptional repressor of mucin biosynthesis. The upstream stimuli of these pathways, including interleukin-4 (IL-4), IL-6, and IL-13, increased dramatically upon exposure to M. pneumoniae. Inhibition of the STAT6, STAT3, and EGFR signaling pathways significantly restored the expression of FOXA2 and attenuated the expression of airway mucins MUC5AC and MUC5B. Collectively, these studies demonstrated that M. pneumoniae induces airway mucus hypersecretion by modulating the STAT/EGFR-FOXA2 signaling pathways.A irway mucus forms a protective coating that entraps foreign particles and microbes, facilitating their clearance by mucociliary transport. Mucus is composed mainly of mucin glycoproteins, water, ions, and cellular debris. Mucins are the major macromolecular component of the mucus gel responsible for its viscoelastic, rheological, and clearance properties. MUC5AC and MUC5B are the major mucins of human airways (1-3). Although a deficient mucous barrier intuitively leaves the lungs vulnerable to injury, aberrant mucin secretion and accumulation contribute significantly to the pathogenesis of airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) (1-4). Mucus plugging in asthmatic and COPD lungs is a major cause of airway narrowing and death (5, 6). Furthermore, hypersecretion of MUC5AC is detrimental during acute lung injury (7).The ability of microbial pathogens to induce mucus secretion suggests that it is one of the mechanisms of infection-induced exacerbation in airway diseases (8-11). M. pneumoniae is the most common cause of community-acquired pneumonia. Moreover, M. pneumoniae has long been recognized as a trigger of both chronic infection and acute exacerbation in multiple chronic airway diseases, including asthma (12, 13). Several virulence mechanisms of M. pneumoniae are known, including cytoadherence through a polar attachment organelle (14), generation of reactive oxygen species (ROS) (15), and secretion of the community-acquired respiratory distress syndrome (CARDS) toxin (16). Alth...

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Temporomandibular joint (TMJ) osteoarthritis is a common chronic degenerative disease of the TMJ. In order to explore its aetiology and pathological mechanism, many animal models and cell models have been constructed to simulate the pathological process of TMJ osteoarthritis. The main pathological features of TMJ osteoarthritis include chondrocyte death, extracellular matrix (ECM) degradation and subchondral bone remodelling. Chondrocyte apoptosis accelerates the destruction of cartilage. However, autophagy has a protective effect on condylar chondrocytes. Degradation of ECM not only changes the properties of cartilage but also affects the phenotype of chondrocytes. The loss of subchondral bone in the early stages of TMJ osteoarthritis plays an aetiological role in the onset of osteoarthritis. In recent years, increasing evidence has suggested that chondrocyte hypertrophy and endochondral angiogenesis promote TMJ osteoarthritis. Hypertrophic chondrocytes secrete many factors that promote cartilage degeneration. These chondrocytes can further differentiate into osteoblasts and osteocytes and accelerate cartilage ossification. Intrachondral angiogenesis and neoneurogenesis are considered to be important triggers of arthralgia in TMJ osteoarthritis. Many molecular signalling pathways in endochondral osteogenesis are responsible for TMJ osteoarthritis. These latest discoveries in TMJ osteoarthritis have further enhanced the understanding of this disease and contributed to the development of molecular therapies. This paper summarizes recent cognition on the pathogenesis of TMJ osteoarthritis, focusing on the role of chondrocyte hypertrophy degeneration and cartilage angiogenesis.

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High-throughput profiling of in situ gene expression represents a major advance towards the systematic understanding of tissue complexity. Applied with enough capture area and high sample throughput it will help to define the spatio-temporal dynamics of gene expression in tissues and organisms. Yet, current technologies have considerable bottlenecks that limit widespread application. Here, we have combined DNA nanoball (DNB) patterned array chips and in situ RNA capture to develop Stereo-seq (Spatio-Temporal Enhanced REsolution Omics-sequencing). This approach allows high sample throughput transcriptomic profiling of histological sections at unprecedented (nanoscale) resolution with areas expandable to centimeter scale, high sensitivity and homogenous capture rate. As proof of principle, we applied Stereo-seq to the adult mouse brain and sagittal sections of E11.5 and E16.5 mouse embryos. Thanks to its unique features and amenability to additional modifications, Stereo-seq can pave the way for the systematic spatially resolved-omics characterization of tissues and organisms.