Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12 h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16 h post fertilization. From 24 h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

The zebrafish spil promoter drives myeloid-specific expression in stable transgenic fish

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.

Using gene-history and expression analyses to assess the involvement of LGI genes in human disorders

Mutations in the leucine-rich, glioma-inactivated 1 gene, LGI1, cause autosomal-dominant lateral temporal lobe epilepsy via unknown mechanisms. LGI1 belongs to a subfamily of leucine-rich repeat genes comprising four members (LGI1–LGI4) in mammals. In this study, both comparative developmental as well as molecular evolutionary methods were applied to investigate the evolution of the LGI gene family and, subsequently, of the functional importance of its different gene members. Our phylogenetic studies suggest that LGI genes evolved early in the vertebrate lineage. Genetic and expression analyses of all five zebrafish lgi genes revealed duplications of lgi1 and lgi2, each resulting in two paralogous gene copies with mostly nonoverlapping expression patterns. Furthermore, all vertebrate LGI1 orthologs experience high levels of purifying selection that argue for an essential role of this gene in neural development or function. The approach of combining expression and selection data used here exemplarily demonstrates that in poorly characterized gene families a framework of evolutionary and expression analyses can identify those genes that are functionally most important and are therefore prime candidates for human disorders.

MicroRNA expression patterns in post-natal mouse skeletal muscle development

BACKGROUND: MiRNAs are essential regulators of skeletal muscle development and homeostasis. To date, the role and regulation of miRNAs in myogenesis have been mostly studied in tissue culture and during embryogenesis. However, little information relating to miRNA regulation during early post-natal skeletal muscle growth in mammals is available. Using a high-throughput miRNA qPCR-based array, followed by stringent statistical and bioinformatics analysis, we describe the expression pattern and putative role of 768 miRNAs in the quadriceps muscle of mice aged 2 days, 2 weeks, 4 weeks and 12 weeks.

RESULTS: Forty-six percent of all measured miRNAs were expressed in mouse quadriceps muscle during the first 12 weeks of life. We report unprecedented changes in miRNA expression levels over time. The expression of a majority of miRNAs significantly decreased with post-natal muscle maturation in vivo. MiRNA clustering identified 2 subsets of miRNAs that are potentially involved in cell proliferation and differentiation, mainly via the regulation of non-muscle specific targets.

CONCLUSION: Collective miRNA expression in mouse quadriceps muscle is subjected to substantial levels of regulation during the first 12 weeks of age. This study identified a new suite of highly conserved miRNAs that are predicted to influence early muscle development. As such it provides novel knowledge pertaining to post-natal myogenesis and muscle regeneration in mammals.

Efficacy of using cancer stem cell markers in isolating and characterizing liver cancer stem cells

Recent evidence suggests that a subset of hepatocellular carcinomas (HCCs) are derived from liver cancer stem cells (LCSCs). In order to isolate and characterize LCSCs, reliable markers that are specific to these cells are required. We evaluated the efficacy of a range of cancer stem cell (CSC) markers in isolating and characterizing LCSCs. We show that the most widely used CSC markers are not specific to LCSCs. By western analysis, protein expression of the common markers showed no significant difference between HCC tumor tissues and adjacent non-cancerous liver. Further, isolation of LCSCs from common HCC cell lines using FACScan and microbeads showed no consistent marker expression pattern. We also show that LCSCs have unique subtypes. Immunohistochemistry of HCC tissues showed that different HCCs express unique combinations of LCSC markers. Quantitative real-time polymerase chain reaction analysis showed that LCSCs isolated using different markers in the same HCC phenotype had different expression profiles. Likewise, LCSCs isolated from different HCC phenotypes with the same marker also had unique expression profiles and displayed varying resistance profiles to Sorafenib. Thus, using a range of commonly used CSC markers in HCCs and cell lines, we demonstrate that currently available markers are not specific for LCSCs. LCSCs have unique subtypes that express distinctive combinations of LCSC markers and altered drug resistance profiles, making their identification problematic.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

Background: ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.
Results: In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals.
Conclusions: In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.

Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia

Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.

Identifying emerging hotel preferences using emerging pattern mining technique

Hotel managers continue to find ways to understand traveler preferences, with the aim of improving their strategic planning, marketing, and product development. Traveler preference is unpredictable for example, hotel guests used to prefer having a telephone in the room, but now favor fast Internet connection. Changes in preference influence the performance of hotel businesses, thus creating the need to identify and address the demands of their guests. Most existing studies focus on current demand attributes and not on emerging ones. Thus, hotel managers may find it difficult to make appropriate decisions in response to changes in travelers' concerns. To address these challenges, this paper adopts Emerging Pattern Mining technique to identify emergent hotel features of interest to international travelers. Data are derived from 118,000 records of online reviews. The methods and findings can help hotel managers gain insights into travelers' interests, enabling the former to gain a better understanding of the rapid changes in tourist preferences.