Drosophila melanogaster. We have performed a computational screen for novel microRNAs in Drosophila melanogaster by searching for phylogenetically conserved putative pre-miRNA structures. The ability of predicted novel miRNA precursors to be processed to produce miRNAs was experimentally verified in S2 cells and in several cases their endogenous expression at was validated by Northern blots. After experimental validation, the predictions were cross-checked with reference to a newly released set of small RNA sequences. Combining both datasets allowed us to identify 53 novel miRNA loci in the fruit fly genome 22 of which we had predicted computationally. This significantly expands the set of known miRNAs in Drosophila melanogaster. Most novel miRNAs contain unique seed sequences not found in other Drosophila miRNAs and are therefore expected to regulate novel sets of target genes. This data provides the basis for future genetic analysis of miRNA function and will aid the discovery of orthologous sequences in other species.MicroRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in post-transcriptional regulation of metazoan development, homeostasis and disease. The full set of miRNAs is not known for any species and it is believed that many await discovery. The recent assembly of 15 insect genomes has provided the opportunity to identify novel miRNAs in the fruit fly,  Subsequently large numbers of miRNAs have been identified by cloning and sequencing of short RNAs isolated from animals, plants and even the unicellular green alga Chlamydomonas reinhardtiiDrosophila miRNAs has been deposited in mirBase (version 10.0) Drosophila melanogaster genome MicroRNAs (miRNAs) are small non-coding RNAs, typically 21–23 nt long, which serve as post-transcriptional regulators of gene expression. The founding members of this class, MicroRNA genes have been found as independent transcription units as well as within introns of protein coding genes. More than half of the known loci are part of tandem arrays within operon-like clusters in vivo studies of loss-of-function phenotypes demonstrate the important regulatory role of miRNAs e.g. in development miRNAs mediate post-transcriptional inhibition by base-pairing with their cognate target mRNAs. Extended complementarity, as is typically found in plants, leads to cleavage of the target. Limited pairing, including but not limited to the 5′ seed region of the microRNA causes translational inhibition, and in some cases increased RNA turnover The majority of miRNAs known to date has been identified through cloning and sequencing of small RNA libraries. Additionally, complementary computational tools for predicting miRNAs have been developed. Two basic strategies have been described to predict novel microRNAs: Sequence similarity searches led to the identification of novel members of previously established miRNA families, many of which are conserved across the animal kingdom Drosophila melanogaster, we have combined a computational screen for generic ncRNAs using RNAz with stringent filtering criteria specifically targeting miRNA precursors. We have validated 22 of the newly predicted loci experimentally. 31 additional loci were identified from recently published small RNA sequence libraries.The publication of many newly sequenced and assembled genomes presents us with an unprecedented wealth of information about evolutionary conservation of coding and non-coding sequences. Based on the observation that the evolution of many non-coding RNAs (ncRNAs) is constrained by functionally important secondary structure elements, novel approaches have been developed to scan whole metazoan genomes for putative ncRNAs. The RNAz program employs a support vector machine (SVM) to evaluate both thermodynamic stability of candidate sequences as well as the conservation of the predicted secondary structure Drosophila melanogaster is a powerful model system to study metazoan development. Its compact genome is well annotated, owing to many years of genetic experimentation Drosophila species, the honeybee (A. mellifera), the flour beetle (T. castaneum) and the mosquito (A. gambiae) has recently become available. To identify novel microRNAs, we took advantage of the whole-genome alignments of 9 and 15 insect species provided by the UCSC genome browser The fruit fly Drosophila melanogaster genome show high conservation across the full sequence of the pre-miRNA, we also included regions of constantly high conservation  in both reading directions (53/56). The predicted hairpins were often symmetrical. These candidates were therefore expressed in both orientations to direct production of primary transcripts from both strands. Processing of these long primary transcripts was assayed by Northern blotting, and detected by hybridization with oligonucleotide probes directed against each arm of the predicted hairpin. Out of 33 candidates tested, 16 gave rise to a distinct small RNA  were identified both computationally and by sequencing. In addition, the small RNA libraries provided evidence for 31 additional loci that we had not predicted. Three predicted miRNAs  could be detected in untransfected S2 cells and in developing embryos by Northern blot analysis , but werDrosophila melanogaster miRNA, mir-12, suggesting that both miRNAs could regulate a similar set of targets  could be verified by Northern blot analysis or sequencing, but were missed in previous studies, demonstrates the power of this approach.The degree of sequence conservation of miRNA precursors between related species varies widely  and 15 (multiz15) insect species were downloaded from the UCSC genome browser. Starting from the “most conserved” track for each alignment generated by the Phastcons program, we padded the genomic coordinates by 25 bps on both sides. Neighboring sequences closer than 50 bps to each other were combined into a single region. All sequences overlapping annotated genes according to the “Flybase genes” and “Refseq genes” tracks of the UCSC genome browser were removed.Input windows were prepared with the rnazWindow.pl script of the RNAz package with default settings. This resulted in alignments with a maximum length of 200 bps  and a minimum number of aligned sequences (>50% identity) from at least 4 different species. RNAz (version 1.0) was used to score the alignments and hits with a probability score p>0.9 were stored. Overlapping predictions were joined using the rnazCluster.pl script of the RNAz package and filtered for hairpin length , thermodynamic stability (z-score <−3.5). The remaining predictions were uploaded to a local installation of the UCSC genome browser and their multiz15 phastcons conservation profile inspected manually.Drosophila embryos using Trizol (Invitrogen) and precipitated with isopropoanol overnight at −20°C. 15 µg of total RNA was separated on a 15% denaturing polyacrylamide gel and blotted using a chemical cross-linking procedure 32P-ATP using T4 polynucleotide kinase (NEB).Amplicons of ca. 0.5 kb length, centered on the predicted pre-miRNA were generated by PCR and cloned into vector pAc5.1B (Invitrogen). S2 cells were grown in the absence of serum, transfected using Cellfectin (Invitrogen) and incubated for 3 days at 25°C. Total RNA was extracted from transfected or control cells, as well as from staged collections of Sequences detected in Drosophila small RNA libraries de novo by moving a 110 bp window (stepsize 5) over all bases recovered at least 10 times. RNAfold miRNA loci were identified from the small RNA libraries Data S1Pdf document containing information about all validated novel miRNAs, including RNAz alignment and structure models.(2.17 MB PDF)Click here for additional data file.Table S1Genome data on 53 new miRNAs. Information about all 53 validated novel miRNAs, including their genomic coordinates (genome version 5) and their mature sequences.(0.02 MB PDF)Click here for additional data file.


However, much of our understanding of airway epithelial cell function in asthma has been derived from ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines  by cultured tracheal explants, was assessed by ELISA.Using a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM) allergen were established to investigate airway mucosal responses The general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h) explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants.in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the application of tracheal explant cultures in relevant ex vivo investigations on the therapeutic and mechanistic modalities of asthmatic disease.Sheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations  Asthma is a complex chronic inflammatory disease, the hallmarks of which include damage to the airway epithelium and underlying parenchyma, recruitment and activation of inflammatory cells, and airflow obstruction associated with remodeling of the airways. The mucosal epithelial layer of the airways plays a pivotal role in the non-specific host defense of the respiratory tract and in shaping both innate and adaptive immune responses of the respiratory system ,2. In asIn vitro cell-based studies have allowed detailed investigations of the molecular mechanisms underlying the pathology of asthmatic disease. This has included studies in humans and appropriate animal models using primary airway epithelial, fibroblast and smooth muscle cells cultured from biopsy samples collected by fibreoptic bronchoscopy or post-mortem tissues [in vitro investigations is the inherent loss of tissue-specific cell differentiation and tissue architecture that is associated with studying isolated cells, and particularly cells of epithelial origin [ex vivo the interactions between epithelium and underlying structural cells of the airway mucosa. Tracheal explants established in sheep display many key features of the in vivo airways such as mucus coverage, mucociliary clearance and cell structure [ex vivo in tracheal explants derived from normal sheep and allergic sheep exposed to house dust mite (HDM) allergen. tissues ,6. Howevl origin ,8. In adtructure . While structure -11 and mtructure , none totructure -15 to inFemale Merino-cross ewe lambs (4-5 months of age) free of significant pulmonary disease  were useDermatophagoides pteronyssinus), and immunized sheep were classed as allergic when they showed increased HDM-specific serum IgE levels as assessed by ELISA [Sheep were sensitized by immunization with solubilized HDM whole extract  pre-soaked in medium  were placed across a 35 mm diameter culture dish sitting within a larger 60 mm dish, to which 3 ml DMEM10 was added. For each treatment, triplicate tracheal biopsies were placed upon the strip of filter paper set up within each plate.Following resection, the trachea was cut longitudinal along the midline through the trachealis, and pinned open onto a dissecting board. The mucosa was dissected gently away from cartilage and small 5 mm discs of tracheal tissue were cut away using a biopsy punch . Tracheal explants were established in culture as outlined previously , with so2/air. In a separate experiment, tracheal tissues were resected from allergic and control (naïve) sheep and tracheal explants established in triplicate cultures for a 24 h period.To investigate possible changes in epithelial morphology and tissue architecture, tracheal biopsies were collected from control (naïve) sheep and explants prepared in triplicate as detailed above. Following the application of 5 μl of DMEM containing HDM allergen (10 μg/ml) or 5 μl of control DMEM to the epithelial surface, explants were cultured for a period of 5 h, 24 h or 48 h at 37°C in 5%COAfter culture, tracheal explants were transferred into a small tube containing 100 μl DMEM10, briefly agitated and (medium washout) samples stored at -20°C for later cytokine analyses. Tissues were then fixed in 4% paraformaldehyde (PFA) and embedded in paraffin for histology. Paraffin-embedded tracheal tissue explants were sectioned (7 μm) and stained with hematoxylin and eosin (H&E), alcian blue/periodic acid-schiff (AB/PAS) or immunostained to identify caspase-3 positive apoptotic cells, mucosal mast cells and eosinophils.2O2, again washed and incubated with 5% normal goat serum (NGS)/2% BSA in PBS-TX. Sections were then incubated with rabbit polyclonal anti-human/mouse activated caspase-3 antibody (Ab) , washed and incubated with biotinylated goat anti-rabbit IgG , and again washed prior to incubation with streptavidin-horseradish peroxidase (HRP) complex . Following washing, sections were developed with 3,3'-diaminobenzidine tetrahydrochloride  and finally dehydrated and cover-slipped with Depex™ tissue mounting fluid .Immunostaining for caspase-3 was used to identify apoptotic cells  in trachFor the detection of mast cells and eosinophils, paraffin embedded tissue sections were blocked as above, then incubated with 10% normal sheep serum in PBS prior to application of primary antibodies. A polyclonal rat anti-ovine tryptase Ab reactive with all ovine respiratory mast cells  was kindIn AB/PAS stained sections, intensely purple staining cells within the epithelium (goblet cells) were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mm length of epithelium.Caspase-3 positive cells, within the epithelial layer only, were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mm length of epithelium.2 area of lamina propria underlying the epithelium. Cell counts for both mast cells and eosinophils were performed on cells that were densely stained. Cells partially stained were classified as degranulated and were not counted.For mast cell and eosinophil cell counts, immunoperoxidase positive cells in the lamina propria underlying the epithelium were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mmTracheal explant washout samples were collected for determination of the ovine cytokines IL-1α, IL-6 and TNF-α by enzyme-linked immunosorbent assay (ELISA), as described previously . The minp < 0.05 was considered significant.Results are presented as means ± SEM. A Kruskal-Wallis non-parametric test was used to compare treatments, within experimental and control groups, and a Dunn's post-hoc test was used where significant. A Mann-Whitney non-parametric test was used for comparisons between experimental and control groups. For all statistical analyses, Priming of the airways with allergen induced a marked recruitment of eosinophils (15-32% of BAL leukocytes) into the BAL fluid when assessed at 48 h post-challenge, similar to that observed in our previous studies ,16.Tracheal tissues were collected from control (naïve) animals to assess the effects of maintaining tracheal explants in culture for 0 h, 5 h, 24 h, and 48 h. Examination of the explants under a dissecting microscope throughout the culture period showed that the tracheal epithelium appeared intact, with evidence of actively beating cilia (not shown). This was supported in the histological examination of tracheal explant tissue sections; while there were several areas showing a less organised arrangement of cells and a reduced number of ciliated cells, there appeared to be little change in epithelial structure and general integrity of the cultured mucosal tissue compared to fresh pre-cultured (0 h) tissue Figure .AB/PAS staining of sections revealed the presence of mucus-producing goblet cells within the tracheal explants. Positively stained goblet cells were seen in all sections, with staining particularly prominent in the tracheal epithelium prior to culture, followed by a dramatic reduction in the level of staining throughout the culture period Figure . Cell coImmunostaining for caspase-3, a marker for cell apoptosis, was examined in paraffin sections of tracheal explants from control animals before and after culture. Caspase-3 staining of apoptotic bodies was seen in all sections of cultured tissue and staining was evident within the epithelium and underlying lamina propria Figure . No posiTracheal tissues were collected from allergic and control (naïve) sheep. The airways of allergic animals had been 'primed' with three aerosolised HDM challenges, resulting in the induction of airway eosinophilia and raised allergen-specific IgE levels in serum and bronchoalveolar lavage (BAL), as noted previously . Two weeAt 0 h there were intense, AB/PAS-stained goblet cells in the epithelium as reported earlier , as they were below the detection limit for these assays.IL-6 secretion was not detected in any of the control tracheal tissues (< 2.65 ng/ml), but was clearly evident in the washout samples from allergic tracheal explants cultured for 24 h in the presence of media alone (4.08 ± 0.67 ng/ml) or with HDM allergen .in vitro models, in particular primary cell cultures, has greatly contributed to our understanding of molecular pathways in allergic inflammation. However, asthma is a complex disease, and in vitro studies that focus on isolated cell populations may not accurately represent in vivo conditions where dynamic and complex interactions take place between different structural and inflammatory cells. Tissue explant cultures established ex vivo have been used for a variety of applications, and while their use may be restricted to short-term studies, they offer a distinct advantage in providing a more complete tissue microenvironment for experimentation that preserves many of the in vivo tissue characteristics. Previous studies in sheep have reported the use of tracheal explants for cystic fibrosis research [ex vivo tracheal explant model as an investigative tool for asthma research.The use of research ,11. The ex vivo) conditions rather than being solely indicative of tissue injury and cell death, the epithelial structure and general morphology of cultured tracheal explants was maintained for up to 48 h in culture. This is in agreement with an earlier study using this model system which reported that explants maintained over this period in culture clearly display cilia and microvilli (as assessed by scanning electron microscopy and light microscopy) as well as ciliary beating, retain normal barrier functions including mucociliary clearance and mucus production, and an intact cellular structure [We demonstrate in the present study that, other than the dramatic loss of goblet cell numbers, which likely reflects a tissue response to the new  challenge . In the Airway eosinophilia is a central component of asthma pathogenesis. Upon recruitment to the airways, eosinophil activation results in the release of mediators and toxic granule proteins, the actions of which include epithelial damage and stimulation of mucus secretion ,27. In pThe secretion of the cytokines IL-6, TNF-α and IL-1α in the tracheal explant cultures was assessed by ELISA, and while TNF-α and IL-1α were undetected, detectable levels of IL-6 were released by allergic tissues but absent in control tissues. A previous study of IL-6 mRNA expression in human airway epithelial cells observed very low or no IL-6 levels in non-asthmatic tissue compared to asthmatic tissue . IL-6 lein vitro models, is the preservation of the tissue architecture and microenvironment that may better reflect conditions and effects encountered in vivo. The present studies suggest this tracheal explant model may be most relevant for investigations over shorter culture periods (< 24 h), since tissue changes became more evident over longer periods in culture. As allergen stimulation causes an immediate hypersensitivity response in asthmatic subjects following degranulation of mast cells and release of inflammatory mediators, it would be interesting to examine in more detail the early time points after HDM challenge of tissue explants and measure the release of these mediators during this period in both allergic and non-allergic explants. Using a validated large animal model with similar airway structures to human, the tracheal explant model offers great potential for studying the complex mechanisms driving allergic disease and could be used as a platform for the screening of potential anti-asthma therapeutics.Animal experimental models have proven to be imperative in understanding disease processes and recent research has focused in particular on the inflammatory mediators in the airways, in the hope of developing more specific and effective therapeutic avenues for asthma treatment. This study examined a tracheal tissue explant model as an investigative tool in asthma research. A distinct advantage of this explant model, compared to other None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.LA carried out the majority of the experimental work and data analysis and drafted the manuscript. RJB developed the culture model and with ENTM conceived the study and participated in the experimental design, data analysis and manuscript preparation. All authors read and approved the final manuscript.


The mammary gland undergoes a sophisticated programme of developmental changes during pregnancy/lactation. However, little is known about processes involving initiation of apoptosis at involution following weaning. We used fur seals as models to study the molecular process of involution as these animals display a unique mammary gland phenotype. Fur seals have long lactation periods whereby mothers cycle between secreting copious quantities of milk for 2 to 3 days suckling pups on land, with trips to sea alone to forage for up to 23 days during which time mammary glands remain active without initiating apoptosis/involution.We show the molecular basis by which alpha-lactalbumin (LALBA), a secreted milk protein, is absent in Cape fur seals and demonstrate an apoptotic function for LALBA when exposed to mammary cells.We propose that apoptosis does not occur in fur seal mammary glands due to lack of LALBA in fur seal milk, allowing evasion of involution during a foraging trip. Our work identifies LALBA as a milk factor that feeds back on the mammary gland to regulate involution. The mammary gland represents one of the most dramatic examples of physiological development. The massive changes of form and function of mammary glands over the life span of a female are characterized by extreme changes in cell proliferation, differentiation, secretion and death, which accompanies pregnancy, lactation and involution upon weaning. While milk is sucked from the mammary gland it provides nutrition and immunity to the young. However, upon milk stasis, due to absence of sucking at weaning, the mammary gland regresses and is remodelled by a process known as involution, which cleanses the gland and returns it to a virgin-like state. Although the mammary gland appears vastly regulated it is also highly susceptible to cancer, with mortality associated with breast cancer rating amongst the highest causes of death for women in the western world.The study of apoptosis in the mammary gland during involution is important for understanding both the normal biology of post-natal regression and the events leading to mammary gland tumorigenesis. Interestingly, some mammals have modified their lactation cycle in order to accommodate and adapt to extreme environmental pressures. Animals such as otariid seals  exhibit an unusual lactation phenotype  which diThe three families of Pinnipeds, comprising Phocids , Odobenids , and Otariids  evolved from a carnivorous ancestor around 25 million years ago and diverged during the middle Miocene (10 million years ago)  whereby In contrast, ancestral otariid seals retained smaller body sizes and insulating fur, and bred at rockeries to gain proximity to local prey resources adopting a 'foraging lactation' strategy . The smaFor other mammals, accumulation of milk in the mammary gland due to cessation of sucking by the young allows factors present within milk to regulate mammary epithelium, causing downregulation of milk protein gene expression, followed by involution via apoptotic cell loss . During LALBA), which normally encodes a milk protein involved in lactose synthesis, has undergone a number of cis-acting mutations, which render the protein absent in otariids. We show that LALBA causes apoptosis of mouse and human mammary epithelial cell lines and fur seal primary mammary cells. We propose that lactose synthesis and mammary gland involution does not normally occur in fur seal mammary glands due to absence of LALBA in fur seal milk and show that previous predictions based solely on isolation of cDNA in otariid mammary glands [Here we show the alpha-lactalbumin gene  during pregnancy, on-shore lactation and off-shore lactation. These results were compared with microarray analysis patterns of genes expressed in human (Homo sapiens), cow (Bos taurus) and mouse (Mus musculus) mammary gland during pregnancy and lactation. Using this approach candidate apoptosis genes were examined for significant differences in expression, and unusually low levels of a major milk protein gene, LALBA, were observed in mammary glands of lactating Cape fur seals.Affymetrix canine arrays were used to examine the pattern of gene expression in mammary tissue from Cape fur seals  and levels of expression of both these milk protein genes increased dramatically with onset of lactation  and Antarctic fur seal  was amplified, sequenced and compared with three phocid seals; grey seal , ringed seal (Pusa hispida) and harbour seal (Phoca vitulina). Analysis of a 718 bp region of the immediate proximal promoter for all seal species showed 96.78 to 97.21% sequence identity, suggesting high level of conservation. However, the TATA box, a conserved DNA sequence which initiates transcription in many genes [LALBA gene in guinea pigs at positions -60 to -57 (relative to the start of transcription) [LALBA TATA box in many other mammalian species comprises the sequence AATAAA, with the exception of mouse and human which comprise CATAAA and TATAAA respectively   was alteLALBA gene expression levels was assayed by use of reporter gene constructs. This was performed in order to determine if low levels of Cape fur seal LALBA transcript detection were due to low levels of LALBA gene transcription or low LALBA transcript stability.A direct comparison between otariid and phocid LALBA promoters and translation initiation codons (ATG) of an otariid seal  and a phocid seal  were ligated into to a luciferase reporter gene. For both species the immediate -718 bp to 3 bp promoter fragment was cloned in front of a luciferase gene within a basic vector (pGL3) and promoter activity was measured by transiently transecting plasmids into a bovine mammary epithelial cell line (BME-UV1) and a human embryonic kidney cell line (HEK293T) and measuring luciferase activity under a variety of conditions. BME-UV1 cells, which have previously been shown to induce expression of milk protein gene promoters in the presence of lactogenic hormones [LALBA expression levels, while HEK293T cells were used to measure LALBA expression in the presence of an introduced enhancer element, measuring the capacity of the TATA box to initiate transcription. In BME-UV1 cells, significant expression levels failed to be detected for both seal species so a STAT5 site was subsequently introduced by in vitro mutagenesis  and induced conditions , compared with the Cape fur seal LALBA reporter constructs (P = 0.038)  alone failed to significantly increase levels of transcription compared with the wild-type Cape fur seal (AAGAAA) promoter construct in the presence of the introduced STAT5 site. Each promoter was also assayed in BME-UV1 cells using an enhancer sequence (pGL3-Enhancer) to elevate basal transcription levels. Again the harbour seal LALBA reporter constructs showed significantly higher levels of expression compared with the Cape fur seal LALBA reporter constructs (P = 0.000) and the in vitro mutated Cape fur seal TATA box (AATAAA) alone failed to significantly increase levels of transcription compared with the wild-type Cape fur seal (AAGAAA) promoter construct  Figure , similarAT5 site . The inft Figure . The LALs Figure .LALBA expression observed occurred at the level of transcription and was not due to low LALBA transcript stability. These results also showed that the Cape fur seal LALBA TATA mutation alone is not responsible for the low level of expression observed  and phocid seals , LALBA genomic fragments were cloned into CMV expression vectors and transfected into HEK293T cells. Genomic fragments comprised all 4 exons and 3 introns with 3'UTR. Following transfection, analysis of LALBA transcripts by RT-PCR and sequence analysis showed the presence of one major otariid LALBA transcript, corresponding to LALBA(s), with the structure exon 1–2–3 and lacking exon 4, and two minor transcripts  with the structure exon 1–2–3, intron 3 exon 4, while the second minor transcript LALBA(Δ) is due to incorrect splicing of intron 1 leading to deletion of the final 60 bp in intron 1. This deletion is caused by use of a 5' splice consensus sequence (AAT/GTGAGCT) within intron 1 and is fused in frame with intron 2, causing an in-frame deletion of 20 amino acids. Analysis of phocid seal  LALBA genomic fragments revealed the presence of one major LALBA transcript corresponding to correctly spliced exons 1, 2, 3 and 4 and a minor transcript corresponding to LALBA(Δ)  Figure . The pres Figure . Expressin silico translations of otariid cDNAs suggested the protein is truncated, removing the final 19 residues [LALBA genomic fragments was tested for presence of secreted LALBA at 24 and 72 hours by western blot using a polyclonal anti-bovine LALBA antibody. Although expression levels of otariid and phocid LALBA transcripts were observed to be equal due to presence of the CMV promoter  LALBA genes  transcript, predicted to be 12.2 kD, was not observed.Previous predictions based on residues . Conditir Figure , and equs Figure . A crudeWe investigated the death-inducing properties of bLALBA in the fur seal mammary gland by assaying LALBA apoptotic potential using Cape fur seal mammary cells (CfsMCs). These cells are isolated from mammary tissue and comprise a population of epithelial cells, myoepithelial cells and fibroblasts that have previously been shown to form mammospheres capable of expressing milk protein genes under induction by lactogenic hormones . bLALBA P < 0.001).In order to determine whether bLALBA was inducing apoptosis, as has been observed in RAW 264.7 cells , we treaIn order to show that bLALBA induces dose-dependent apoptotic responses in other mammary epithelial cell lines, bLALBA was added in increasing concentrations to mouse mammary epithelial cells, HC11, and to the human breast cancer cell line MCF-7. Similar to CfsMCs, higher doses of bLALBA caused all cells to die, while lower does allowed survival of a small population of cells which went on to proliferate Figure  to 8D. MThe time required for bLALBA to induce an apoptotic response was also examined by exposing cells to bLALBA for 1 and 8 hours and then removing bLALBA and replacing with fresh media. These experiments showed apoptotic effects of bLALBA were observed when cells were exposed to 8 hours of bLALBA and then removed, but apoptosis was not observed when cells were exposed for 1 hour and then removed Figure .Specific triggers for the process of mammary involution are unknown but autocrine feedback mechanisms have been proposed . The souLALBA mRNA during the lactation cycle compared with expression levels in other mammals, which show LALBA is one of the most highly expressed genes in the mammary gland during lactation [It has long been established that LALBA, a major milk component, plays a central role in the mammary gland as the regulatory subunit of lactose synthase . We showactation . LALBA hactation  and RAW2actation . LALBA wLALBA expression levels between otariid and phocid seals showed otariid  LALBA expression levels were very low and suggested a negligible amount of transcription occurs from this promoter. The low level of otariid LALBA transcription was predicted to be due to an altered TATA box in otariid seals. All three otariid species displayed a T-G transversion within the third position of the TATA box creating an AAGAAA sequence. This substitution is predicted to lead to poor binding of the TATA binding protein and RNA polymerase which are necessary for transcriptional activity [LALBA promoter failed to increase levels of transcription, suggesting that another/other mutations also play a role in preventing successful transcription of LALBA. Like most pseudogenes where evolutionary pressures are no longer required to prevent divergence, the otariid LALBA has likely undergone a number of promoter mutations, which are conserved in other lineages where LALBA is functional.Comparison of activity . The conactivity . CorrectLALBA transcript abundance showed one major transcript, LALBA(s) was generated for all otariid species, with LALBA(l) and LALAB(Δ) detected as minor transcripts. Interestingly, LALAB(Δ) was also detected in phocid species. Other species such as dogs, humans, sheep, rats, mice and pigs also have the same cryptic 5' splice sequence in exon 1, suggesting the presence of LALAB(Δ) in species other than seals.Analysis of LALBA transcripts showed otariid LALBA mRNA failed to be translated into secreted protein. These data are consistent with earlier studies, which have failed to detect LALBA protein in milk of otariid species [Macropus eugenii) milk, carbohydrate is low and lactose is absent throughout peak lactation [Translation of otariid and phocid  species ,27. Thes species ) are inc species . These t species  and exce species . Therefoactation , during actation .LALBA in HC11 cells without observing apoptosis of the host cell. Although expression of the transgene was observed to be high due to the presence of a CMV promoter, the amount of protein collected in the conditioned medium was determined to be less than that required for effective LALBA-induced apoptosis. In addition, stably transfected cells have been derived which express bLALBA which are also not detrimentally affected [Evidence presented here shows that bLALBA causes apoptosis of mouse and human mammary epithelial cell lines and fur seal primary mammary cells, demonstrating that although LALBA is absent in fur seal milk, the LALBA-mediated apoptotic pathway is still intact in fur seal mammary cells. It is interesting to note that all cell types in the Cape fur seal mammary population responded to bLALBA, suggesting that the apoptotic response to LALBA is not limited to epithelial cells, but also affects other cells types such as fibroblast and myoepithelial cells. The loss of LALBA apoptotic activity by heat treatment confirms the active component is of protein origin and suggests the apoptotic effects observed do not occur when the protein is fragmented by digestion with pepsin. Previously a multimeric form of LALBA (MAL), isolated from the casein fraction of human milk and formed by low pH treatment and ion-exchange, has been shown to reduce cell viability in kidney, intestine, bladder, lung cell lines, lymphocytes and thymocytes, but healthy cells were not affected  and mammaffected ; we suggin vivo epithelium. It could be postulated that LALBA acts only at a critical concentration and requires a specific amount of time in contact with the cell, or undergoes a conformational change during milk stasis in order to elicit its apoptotic potential. Involution only occurs following milk stasis and it has been previously demonstrated that LALBA concentrations increase in milk during mammary gland involution, while other milk proteins show decreases in concentration [in vivo epithelium during lactation, as seen by decreased milk production in cows as lactation proceeds [Our data suggest that low levels of LALBA may not cause apoptosis in the ntration . LALBA mproceeds . This grWe suggest that exposure of mammary epithelial cells to LALBA may be a mechanism for mammary gland involution during milk stasis at weaning, a time when LALBA levels increase in the milk. To support this we have presented a molecular analysis of a relevant lactation model, the fur seal, which avoids involution in the presence of milk stasis and showed that these animals do not produce LALBA in their milk. In addition we have shown that mammary epithelial cells, when exposed to similar LALBA concentrations to those found in milk, undergo an apoptotic response.LALBA-deficient mice [LALBA-deficient mice [LALBA-deficient mouse mammary glands were distended and so engorged with milk that tight junctions were compromised and milk was observed to move from the alveolar space and enter into the surrounding mammary tissue. However, there was no evidence of apoptosis or involution which would normally occur by this time in the absence of milk removal [LALBA-deficient mouse is consistent with absence of apoptosis and involution in the LALBA-deficient otariid seal.We propose that mammary gland involution does not normally occur in fur seal mammary glands due to absence of LALBA in fur seal milk and not due to the presence of a truncated LALBA protein as previously reported . The absent mice . Milk ofent mice  is high  removal . The absLALBA phenotype. As discussed previously, wallaby milk during peak lactation is devoid of lactose; however, LALBA is secreted in wallaby milk at similar levels to other mammals throughout lactation [LALBA in the current study showed similar levels to other mammals such as pig and mice during peak lactation. This shows that wallabies have uncoupled secretion of LALBA and lactose production. In contrast to fur seals, wallabies do not have an interrupted pattern of lactation and do not have long periods of non-suckling activity, so we predict that continued secretion of LALBA during wallaby lactation is required solely for rapid initiation of mammary gland involution at weaning.The tammar wallaby is another model species with an unusual actation . ExaminaHere we show that LALBA acts an as apoptotic inducer of mammary epithelial cells and we correlate the lack of LALBA with the absence of apoptosis in fur seal mammary glands. This study has shown how lack of LALBA has had important evolutionary consequences leading to the phenotypic trait of foraging lactation in fur seals that is different from the fasting lactation in phocid seals, and by doing so identifies LALBA as a likely candidate trigger for involution in the mammary gland.RNA was isolated  from Cape fur seal, wallaby, pig and mouse mammary tissue or from cells derived from California sea lion and Antarctic fur seal milk during peak lactation, human milk and colostrum at 24 hours prior to birth and at 3 and 4 months peak lactation, or HEK293T cells. mRNA purification was performed by NucleoSpin kit (BD Biosciences).n = 2), on-shore lactation (n = 2) and offshore foraging (n = 1) or human during pregnancy (n = 2) and lactation (n = 2) was used for gene profiling analysis. Labelling of RNA and microarray processing was contracted to the Australian Genome Research Facility  using Canine genome 2.0 or Human genome Affymetrix genechips. Cow Affymetrix results were kindly provided by Dr PA Sheehy, Faculty of Veterinary Science, University of Sydney, NSW, Australia. Data was normalized using RMA methods in Bioconductor [[RNA isolated from Cape fur seal mammary glands during pregnancy (onductor . Mouse Anductor [.mRNA (1 μg) was used to construct a cDNA library using Creator SMART cDNA Library Construction Kit (BD Biosciences). A total of 11,232 ESTs were sequenced by the Australian Genome Research Facility, Brisbane, Australia.Total RNA 0.1 μg) was used to create cDNA. 0.2 μl cDNA was used in PCR with 20, 25 and 30 cycles of amplification to determine the linear range. Each PCR was performed at least twice. See additional file  μg was uLALBA cDNA probe (550 bp).mRNA (1 μg) was electrophoresed in 1.2% agarose gels, blotted to Zeta Probe and hybridized with a 32P-labelled Cape fur seal NotI and XhoI restriction sites flanking the LALBA fragment and were cloned into pcDNA3.1 (Promega) and sequenced. Both vectors contain CMV promoters and all constructs contained a Kozak sequence A/GTGATTATGA. Cape fur seal, Antarctic fur seal, and harbour seal LALBA were cloned into pTarget, while ringed seal and sea lion DNA were cloned into pcDNA 3.1.DNA was extracted from Cape fur seal liver and California sea lion and Antarctic fur seal milk. DNA from ringed seal, grey seal and harbour seal were gifts from Dr P Johnson , Dr M Walton  and Dr N Lehman , respectively. LALBA coding sequences were amplified using specific primers spanning the ATG of exon 1 and 3' UTR  and sequenced. Mutagenesis of TATA box and STAT5 sites was performed by PCR and sequenced. Plasmids were digested with NcoI/SacI using sites within the vector and 718 bp and 398 bp fragments were subcloned into pGL3-basic and pGL3-enhancer respectively and sequenced. See additional file 7 cells/ml.Mammary tissue was obtained from one Cape fur seal, which was confirmed to be pregnant and non-lactating from inspection of the uterus and mammary glands, respectively. Tissue was immediately transferred to 1× Hanks' Balanced Salt Solution (HBSS)  with 10 μl/ml penicillin/streptomycin  and 2.5 μg/ml Fungizone  on ice and transported back to the laboratory for enzymatic digestion to harvest mammary epithelial cells. Mammary tissue was dissected free from fat, weighed, sliced finely and digested with collagenase and hyaluronidase (25 g tissue per 100 ml media) at 37°C for 4 hours. Cells were harvested by filtration . The suspension was centrifuged 80 g/5 minutes, pellets were washed twice with HBSS. Cell suspensions were centrifuged, resuspended in M199 and 90% FCS/10% DMSO , and frozen at a density of ~2 × 102 culture flasks in 5 ml or 10 ml respectively of M199/Hams/Hepes media with 1 μg/ml cortisol, 10 ng/ml EGF, 1 μg/ml insulin supplemented with 20% horse serum and 5% foetal bovine serum. The cells were passaged (up to three times) when confluent using 0.1% trypsin-versine solution in phosphate buffered saline .Cape fur seal mammary cells were cultured in either 25 or 80 cmHEK293T and MCF-7 were grown in DMEM/10% FCS, HC11 cells were grown in M199/HAMS/HEPES/10% FCS, CfsMCs were grown in M199/HAMS/HEPES/20% horse serum/5% FCS with 1 μg/ml cortisol, 10 ng/ml EGF, 1 μg/ml insulin (I) and BME-UV1 cells were grown in 50% DMEM/30% RMPI/20% NCTC-135/5% FCS with 1.0% lactose, 0.1% lactalbumin hydrolysate, 1.2 mM glutathione, 5 μg transferrin/ml, 10 μg L-ascorbic acid/ml. For induction, BME-UV1 media was supplemented with I (1 μg/ml), prolactin (P) (1 μg/ml), dexamethazone (Dex) (1 μM).LALBA transcript and protein analysis media was changed to DMEM/2% FCS and incubated for 72 hours. Media was collected at 24 and 72 hours. After 72 hours RNA was isolated.Prior to transfection BME-UV1 cells were induced for 24 hours with I or I, P, Dex. Transfection was performed using lipofectamine (Invitrogen). Reporter constructs were co-transfected with β-glycosidase vector. Induction or growth media was replaced after 6 hours and incubated for 2 days. For Luciferase assays were performed using Luciferase Assay reagent  and β-glycosidase assays were performed according to the β-galactosidase Enzyme and Assay System with Reporter Lysis Buffer Kit . Luciferase expression values were normalized by comparison with β-glycosidase activity. Each experiment was performed in duplicate and repeated at least twice.2O and assayed for protein by BCA assay . 300 μg protein (~50 μl) was separated on 15% SDS-PAGE gel and transferred to PVDF membrane. Coomassie staining of the gel confirmed an equal amount of protein was loaded per sample. Immunoblotting was conducted by using rabbit polyclonal antibodies specific for LALBA, detected with a goat anti-rabbit HRP-conjugated antibody (Pierce), and visualized using SuperSignal chemiluminescent substrate . The experiment was performed in triplicate using two time points per sample.1 ml of conditioned media (24 and 72 hours) was washed free from salts by use of Amicon ultra 1 kD columns (Millipore). Supernatants were concentrated and resuspended in 50 μl dHCells were plated (2000 cells/well) in 96-well plates in growth media supplemented with 10% FCS. After one day bLALBA  was added and cells were grown for 2, 4, 6, 8, 10, 12 and 14 days before fixing and staining with Sulforhodamine B. Cells were visualized by phase contrast microscopy on corresponding days using an Olympus BX40 microscope and photographed using a DigitalSight DSL1 (Nikon) camera. Each experiment was performed on three separate occasions in quadruplicate. Heat treatment was performed at 95°C for 10 minutes. Pepsin digestion was performed with 1.5 mg bLALBA in HCl (pH 2) 1:500 enzyme to substrate for 60, 90 or 120 minutes/37°C. Products were purified using 1 kD Centrifugal device (Amicon Ultra). Products of digestion were analyzed on SDS-page gels.5 cells per glass chamber slide) were plated and allowed to grow overnight in growth media. The following day media was removed and cells were treated in duplicate with 1 mg/ml bLALBA in growth media for 1, 2 and 4 hours or 0 mg/ml bLALBA. Apoptotic cells were detected using Apotag Peroxidase in situ Apoptosis Detection kit , according to the manufacturer's instructions for staining of cultured cells. Slides were counterstained with 0.5% methyl green and mounted using aqueous mounting medium. The apoptotic cells (brown staining) were viewed and counted using an Olympus BX50 microscope for light microscopy. The apoptotic index was defined by the percentage of brown (dark) cells among the total number of cells in each sample. For each treatment five to eight random fields were used to count apoptotic and live cells. A total of 711 cells (0 mg/ml LALBA), 587 cells (1 mg/ml bLALBA/1 hour), 473 (1 mg/ml bLALBA/2 hours) and 434 cells (1 mg/ml bLALBA/4 hours) were counted for each treatment. Results are represented as percentage of apoptotic and live cells.Mouse mammary epithelial cells, HC11, (1 × 10t-test was used. Each test incorporated data from two independent experiments performed in duplicate on separate days.For expression analysis unpaired student t-tests using 6 degrees of freedom were used to determine statistical significance. For apoptotic analysis a 2-tailed student JAS constructed cDNA library, performed expression/promoter and translation analysis, proliferation assays, apoptotic assay, morphological analysis and drafted the manuscript. CL performed bioinformatics for annotation of sequences. KRN conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.TableClick here for file


Secretary component (SC) is expressed basolaterally as a transmembrane protein (pIg receptor) on secretory epithelial cells. As pIg receptor it plays a central role in humoral immunity by mediating the external translocation of dimeric IgA and pentameric IgM. A few case reports have suggested that reduced or absent SC protein expression is associated with diarrhoeal disease, but there is no convincing evidence that a primary pIg receptor deficiency can occur. In this study the relative presence of SC mRNA was determined by Northern blot analysis and related to immunohistochemically determined SC protein expression in 33 colorectal adenomas (31 patients) with increased risk of developing sporadic colorectal cancer, as well as in 19 colorectal carcinomas from 19 patients with such sporadic tumours. In the adenomas, SC mRNA levels were positively related to SC protein expression; both mRNA and SC protein were negatively related to histological grade. Similarly, SC mRNA levels tended to be related to the SC protein expression in the carcinomas. SC mRNA was detected in all adenomas, and only two of ten carcinomas (10.5%) deemed to be SC deficient by immunohistochemistry also lacked SC mRNA expression, suggesting diallelic alterations in the SC-encoding gene (locus PIGR). This possibility agreed with Southern blot analysis performed on a separate sample of 32 other colonic carcinomas in which the diallelic loss of D1S58  was calculated to be 6.4%. Together these findings suggested that reduced SC protein expression in colorectal adenomas might be a transcriptional defect reflecting the degree of cellular dysplasia, whereas absent SC protein expression in colorectal carcinomas might also involve post-transcriptional defects and occasional diallelic gene deletions representing late events in carcinogenesis.


To determine whether optic nerve head (ONH) astrocytes, a key cellular component of glaucomatous neuropathy, exhibit differential gene expression in primary cultures of astrocytes from normal African American (AA) donors compared to astrocytes from normal Caucasian American (CA) donors.We used oligonucleotide Affymetrix microarray (HG U133A & HG U133A 2.0 chips) to compare gene expression levels in cultured ONH astrocytes from twelve CA and twelve AA normal age matched donor eyes. Chips were normalized with Robust Microarray Analysis (RMA) in R using Bioconductor. Significant differential gene expression levels were detected using mixed effects modeling and Statistical Analysis of Microarray (SAM). Functional analysis and Gene Ontology were used to classify differentially expressed genes. Differential gene expression was validated by quantitative real time RT-PCR. Protein levels were detected by Western blots and ELISA. Cell adhesion and migration assays tested physiological responses. Glutathione (GSH) assay detected levels of intracellular GSH.Multiple analyses selected 87 genes differentially expressed between normal AA and CA (P<0.01). The most relevant genes expressed in AA were categorized by function, including: signal transduction, response to stress, ECM genes, migration and cell adhesion.These data show that normal astrocytes from AA and CA normal donors display distinct expression profiles that impact astrocyte functions in the ONH. Our data suggests that differences in gene expression in ONH astrocytes may be specific to the development and/or progression of glaucoma in AA. Primary open angle glaucoma (POAG), the most common form of glaucoma, is a blinding disease that affects older adults Astrocytes, the major glial cell type in the ONH in humans, provide cellular support function to the axons while interfacing between connective tissue surfaces and surrounding blood vessels JAMA and Genome Biologymyocilin , optineurin , and WD repeat-domain 36 , none of these genes have been found to associate with POAG in African Americans, Afro Caribbeans or in West African populations In this study, we referred to African American individuals (AA) as Black Americans of African ancestry and to Caucasian Americans individuals (CA) as White Americans of Western European ancestry. We used the race, gender and age identification provided with the anonymous donor history according to guidelines published in We have investigated possible differences in ONH astrocytes from populations with different genetic backgrounds, using well characterized primary human astrocytes cultures. We have found that astrocytes derived from normal AA donors exhibit differential gene expression profiles compared to astrocytes derived from normal age-matched CA donors. Our data shows that genes associated with oxidative stress, astrocyte motility, ECM structure, immune responses and the reactive astrocyte phenotype are differentially expressed in normal astrocytes from these different populations. These results demonstrate baseline differences in ONH astrocytes from human populations with different genetic backgrounds and provide a molecular framework for future analyses from normal and glaucomatous astrocytes from different populations.in vivo and in vitroWe established primary cultures of ONH astrocytes from 16 normal African American donors (age 60±11) and 21 normal Caucasian American donors (age 62±12) as described in There were no significantly different demographic variables between populations of the 24 astrocyte lines used in microarray . We analTo better demonstrate the process of identifying significant genes, To classify differentially expressed genes, we separated the genes from RMA-SAM analysis manually by function using available data from public databases . The complete list of the functional categories is shown in RNA from 13 AA and 17 CA age-matched normal donors was used to validate microarray data. Twenty-six genes were selected from 10 functional groups from the RMA-SAM analysis and from the GO lists. 18S was used to normalize the expression value. Fold change obtained by qRT-PCR correlated well with the direction of fold change obtained from the normalized intensity data, confirming the validity of the microarray gene expression patterns .Genes that are associated with intracellular signaling were differentially expressed in AA astrocytes, including cAMP signaling, intracellular vesicular transport, G protein regulation and protein phosphatases . DiffereExpression of the RGS5 gene was upregulated in AA astrocytes by microarray and confirmed by qRT-PCR . The proAmongst genes differentially regulated in AA astrocytes were several genes that impact upon cAMP signaling. β-adrenergic receptor kinase (ADRBK2) is downregulated in AA normal astrocytes . The AK3in vitro , myosin light chain kinase (MYLK) a calcium/calmodulin dependent kinase and PPP1R12B , a myosin phosphatase were upregulated in AA astrocytes compared to CA astrocytes .Western blots detected two isoforms of MYLK: 210 kDa and 130 kDa. The 130 kDa isoform was the predominant form in both AA and CA normal astrocytes. The protein level of MYLK 130 kDa was significantly higher in AA astrocytes compared to CA astrocytes . 210 kDaBased on the gene expression and protein data for MYLK, we compared migration in AA and CA astrocytes. Experiments using a chemotaxis model indicated that AA astrocytes migrated faster than CA . Since Ain vitro  mRNA which was confirmed by qRT-PCR and immunoblot in AA astrocytes . LTBP1, in vitro . MFAP2, in vitro . Howeverin vitro . COL18A1in vitro .Several genes associated with basement membranes were downregulated in AA astrocytes ; includiGenes involved in GSH metabolism, including glutathione S-transferases (GSTs) and gamma-glutamyltransferases (GGTs), are upregulated in AA astrocytes . We measSeveral chaperones were upregulated in AA astrocytes compared to CA astrocytes, including heat shock protein 70 protein 2 (HSPA2), alpha-crystallin-related heat shock protein B6 (HSPB6), and crystallin-β B2 (CRYBB2) . HSP70 pAmong the upregulated genes by microarray in AA astrocytes compared to CA astrocytes were two members of the IGFBP family, IGFBP3 and IGFBP5, the GDNF family receptor α1, the receptor for glial derived growth factor-1 and neurturin . Real tiTo verify the expression of growth factors and cytokines, we used ELISA to measure the quantity of both secreted (medium) and intracellular (cell lysate) forms in 6 AA and 7 CA age matched astrocyte lines. Consistent with lower mRNA levels in AA astrocytes , the totin vitro, we hypothesize that there are in vivo differences in ONH astrocytes in these normal populations relating to cell adhesion/migration, intracellular signaling, extracellular matrix assembly, responses to oxidative stress, innate immune responses and neuronal survival. Population based differences in astrocyte functions may produce differential susceptibility of the ONH to elevated IOP and contribute to the pathophysiology of glaucomatous optic neuropathy in AA, in whom the disease occurs more frequently, has a younger age of onset, is more difficult to treat and progresses more rapidly compared to glaucomatous optic neuropathy in CA.Astrocytes are the major cell type that controls the homeostasis and microenvironment of the retinal ganglion cells axons as they traverse the optic nerve head. The complex interplay of astrocytes with RGC axons involves cell-cell signaling, synthesis of ECM, control of ions and pH, inter- and intracellular transport, immune surveillance, synthesis of growth factors and cytokines, and many other interactions. This study presents data that demonstrate population based differences (AA compared to CA) that may affect important astrocyte functions in the normal ONH. Based on data from gene expression, protein levels and functional assays in vivoin situ. We believe that the findings presented here using in vitro techniques identify functional parameters that should be investigated in vivo.Previous work in our laboratory demonstrated that ONH astrocytes in culture retain many of the phenotypic characteristics that ONH astrocytes display Amongst genes differentially regulated in AA astrocytes were several genes that impact cAMP signaling. β-adrenergic receptor kinase (ADRBK2), which is downregulated in AA normal astrocytes, specifically phosphorylates the agonist-occupied form of the β-adrenergic receptor and promotes its desensitization and internalization AA astrocytes exhibited differential expression of genes associated with cell adhesion and cell migration. Cell adhesion genes which were downregulated in AA astrocytes included: integrin α-6 (ITGA6), which connects these cells with laminin family members in vivo by AA astrocytes may differ than that of CA astrocytes.Upregulated genes in AA astrocytes that are involved in cell adhesion of astrocytes included: GPR56, that activates pathways that inhibit cell adhesion Cell migration involves changes in adhesion and cytoskeletal proteins and changes in cell shape during movement via reorganization of actin filament networks in the cell periphery Using microarray analysis, we have found changes in the expression of genes that suggest the machinery for migration is enhanced in AA astrocytes. In AA astrocytes, these changes included: upregulation of MYLK, a calcium/calmodulin dependent enzyme which phosphorylates myosin regulatory light chains to facilitate myosin interaction with actin filaments, producing contractile activity MYLK genetic variants confer increased risk of sepsis and sepsis-associated with acute lung injury and a more severe asthma phenotype in individuals of African ancestry In vivo, quiescent astrocytes are terminally differentiated cells that exhibit strong and stable attachments to the ECM and neighboring astrocytes. Under conditions of stress, injury and disease, reactive astrocytes become migratory and detach from the underlying ECM. Our microarray data plus those of the migration assay obtained from primary astrocyte cultures strongly suggest that normal AA astrocytes in situ have the potential of becoming migratory cells in response to stress such elevated IOP as in glaucoma.Our microarray data suggests that alterations in elastic fibers and associated microfibrils, a major component of the ECM in the lamina cribrosa, may be a susceptibility factor predisposing the remodeling of the ONH in response to elevated IOP in glaucoma in the AA population. Microarray analysis indicated a significant upregulation in AA astrocytes of elastin (ELN), LTBP1, a member of the elastin microfibrils that binds TGF-β, and COL18A1, a collagen with strong anti-angiogenic properties. MFAP2, the gene encoding for MAGP1, a component of the elastin associated microfibrils, and the gene encoding for LOXL2, a lysyl oxidase that participates in maturation of elastic fiber, were downregulated in AA astrocytes. We previously reported that normal AA astrocytes expressed high levels of elastin mRNA and protein, and decreased levels of LOXL2 LTBP1 is an ECM glycoprotein that plays a major role in storage of latent TGF-β in the ECM and regulate its availability in situ for AA astrocytes.Additional ECM related genes that were downregulated included: nidogen 2 (NID2), a linker protein that joins laminin and collagen IV networks in basement membranes Taken together, population based differences in important components of the ECM that provide elasticity and resiliency to the lamina cribrosa, and in basement membranes that participate in tissue homeostasis and adhesion of astrocytes in the ONH, may render the tissue more susceptible to the elevation of IOP that occurs in glaucoma.In normal AA astrocytes the GDNF family receptor α 1 (GDRA1) was upregulated. GDRA1 is the receptor for Glial -derived neurotrophic factor (GDNF) and neurturin (NTN) which are two potent neurotrophic factors that play key roles in the control of neuron survival and differentiation Two important members of the IGF axis, the insulin like growth factor binding protein 5 and 3 (IGFBP5 and IGFBP3) were upregulated in normal AA astrocytes. IGFBP5 interacts with heparin containing glycosaminoglycans (GAGs) in the ECM and facilitates migration, an important differentiated function of normal AA astrocytes compared to CA The membrane associated heparin binding EGF (HBEGF) was downregulated in AA astrocytes. Several ligands and receptors of the EGF family are known to be expressed in ONH astrocytes including HBEGF in vitroAA astrocytes exhibit downregulation of expression of IL-6, a key cytokine upregulated in reactive astrocytes that promotes glial scar formation and is an impediment to axon regeneration and neuronal survival in the CNS Major histocompatibility complex (MHC) Class I genes  and other molecules in the Class I antigen presentation pathway, such the immunoproteasome , were marginally up-regulated in AA astrocytes. HLA-A and HLA-B belong to the classical class I genes and are expressed in astrocytes in the CNS in disease Astrocytes in the CNS participate in the innate immune response by modulating local reactions to endogenous or exogenous antigens, and by modulating astrogliosis through release of cytokines and by isolating areas of inflammation The antioxidant glutathione (GSH) is vital for cellular defense against oxidative stress in astrocytes and neurons in vitro and perhaps in vivo. Upregulation of GGTs leads to increase in GSH cycling and thus GSH synthesis. The fact that GSH level is siginificantly lower in AA astrocytes, despite higher level of GGTs, suggesting there may be a compromised oxidation-reduction system or a deficient antioxidant response. Oxidative stress may be an important process in glaucomatous optic neuropathy Upregulation of GSTs in AA astrocytes may indicate active detoxification activity by GSH metabolizing enzymes, resulting lower levels of GSH as we demonstrated in vivo or represent subtle genetic changes in the AA population. Thus, differential gene expression amongst AA and CA astrocytes may provide a basis for higher risk for developing glaucoma in AA. Current studies in ONH astrocytes from glaucomatous donors further supports our hypothesis that the gene expression profile of normal AA astrocytes anticipates the changes seen in reactive astrocytes in glaucomatous optic neuropathy.Our study raises many questions that will require future investigations. Our data indicates that the microenvironment supported by astrocytes in the normal ONH in AA has important differences that may impact susceptibility to glaucomatous optic neuropathy. An important question that remains is whether the observed differential gene expressions in AA astrocytes compared to CA astrocytes are the result of continued exposure to stress signals Twenty one human eyes from 21 normal age-matched Caucasian American (CA) donors (age 62±12) and 16 human eyes from 16 normal African Americans (AA) (age 60±11) were used in this study to generate primary cultures of optic nerve head (ONH) astrocytes .Donors did not have history of eye disease, diabetes, or chronic CNS disease. Eyes were obtained from the local eye banks and from the National Disease Research Interchange (NDRI) . Eyes we2 Primaria tissue culture flasks . Explants were maintained in DMEM/F-12 supplemented with 10% FBS  and 10 µl/ml of PSFM . Cells were kept in a 37°C, 5% CO2 incubator. After 2–4 weeks, primary cultures were purified by using modified immunopanning procedure described by Mi and Barres (1999) Cultures of human ONH astrocytes were generated as previously described in vitro transcription was carried out by using the cleaned double-stranded cDNA as a template in the presence of biotinylated UTP and CTP. Purified biotin-labeled cRNA was fragmented before the hybridization. Hybridization of the labeled cRNA to Human Genome HG U133A and HG U133A 2.0 chips  was carried out by using Genechip Instrument System (Affymetrix) at the Genechip Core Facility of Washington University . The arrays were washed and stained with streptavidin-phycoerythrin  followed by scanning with an Agilent GeneArray Scanner G2500A .Normal human eyes used for microarray were from 12 CA normal donors (age 58±11) and 12 AA normal donors (age 58±12) . Total RThe first step in the analysis of the microarray data was to determine which genes were to be considered “present” or “absent.” We estimated the probe-set present/absent calls by using the Wilcoxon signed rank-based algorithm We used RMA and Bioconductor in R To assess the biological significance of the gene list derived above we classified differentially expressed genes in normal AA and CA by manual separation by function using available data from public databases such as UniGene, OMIM and Entrez PubMed and the gene lists from RMA/SAM.In addition, we used GOstats Bioconductor package Independent confirmation of differential expression was conducted using 17 astrocyte cultures from CA and 13 astrocyte cultures from AA age-matched donors obtained as described earlier in Human ONH astrocytes were grown until reaching 90% confluence and Western blot analysis was carried out as previously described ELISA was used to determine the content of HB-EGF and IL-6, using ELISA kits (R & D Systems) specific for each protein. Both cell lysate and culture medium were measured. When cells reached confluence, they were washed with PBS before changed into serum-free DMEM/F-12 medium. After 24 hours of incubation, medium was collected, clarified by centrifugation, and concentrated using Centriprep YM-10 (Millipore) with a molecular weight cut-off of 10,000 kDa. As a control, fresh medium was collected and concentrated in the same fashion. Cells were collected and lysed in 100 µl of 50 mM phosphate buffer with sonication. Five µl of the supernatant was used to measure protein concentration using the Pierce Protein Assay Kit (BCA method). Assay was carried out according to manufacture's protocol. The content for each sample was calculated based on the standard curve, and results were expressed as content (nmol) per mg protein. Six normal AA and seven CA samples were used. The means of the content were considered significantly different if p<0.05 (unpaired t-test).2 for 90 minutes. The cells were then washed 4 times with 250 µl of PBS. 200 µl of cell stain solution (Cell Biolabs) was then added to each well and incubated for 10 minutes at room temperature. The stain was then removed and the cells were washed four times with 500 µl deionized water. After allowing the cells to air dry for 25 minutes, 200 µl of extraction solution (Cell Biolabs) was added to each well and placed on an orbital shaker for 10 minutes. Extracted samples' absorbance was measured at 560 nm by a Thermo Multiskan Spectrum plate reader. Results were corrected by subtracting absorbance of the negative controls. The means from AA and CA groups were considered significantly different if p<0.05 (unpaired t-test).Adhesion was measured in six CA samples and four AA samples using CytoSelect™ Cell Adhesion Assay (Cell Biolabs). Cells were harvested and resuspended in serum-free DMEM/F-12 medium. For each donor, four Collagen IV coated wells and one BSA coated well were used. Fifty thousand cells were added to each well in a volume of 150 µl. For each donor, one Collagen IV well was treated with sterile serum-free DMEM F12 as a negative control. The plate was then incubated at 37°C, 5% CO2 for 24 hours. After removing the media from the inserts, non-migratory cells from the interior of the inserts were removed with cotton-tipped swabs. The inserts were stained with 400 µl of cell staining solution and washed three times with water before transferred to a clean well with 200 µl of extraction solution. The absorbance of the extracted samples was measured at 560 nm by a Thermo Multiskan Spectrum plate reader. The means from AA and CA groups were considered significantly different if p<0.05 (unpaired t-test). To inhibit MYLK, 10 µM of ML-7 (EMD Biosciences) Migratory properties of five CA samples and six AA samples were measured by CytoSelect™ 24-well cell migration assay (Cell BioLabs). Assay was performed according to the manufacture's protocol. Five hundred µl of media supplemented with 10% fetal bovine serum was added to the bottom wells. Fifty thousand cells resuspended in 300 µl serum-free medium were added to the inserts. The plate was then incubated at 37°C, 5% COTotal GSH content was quantified in astrocyte cell lysates based on previously described methods using the Glutathione Assay Kit from Cayman Chemical  Six eyes from normal Caucasian donors (CA) and six eyes from normal age-matched African American (AA) donors were used. Detail methods are described in Primary ONH astrocytes from six normal AA donors and from six normal CA donors were used. Detail methods are described in Table S1Demographic information of normal donors. Demographic information of CA and AA normal donor eyes used to generate primary cultures of ONH astrocytes.(0.09 MB DOC)Click here for additional data file.Table S2Genes differentially expressed in ONH astrocytes from normal AA donors compared to their caucasian counterpart using RMA SAM. List of genes differentially expressed in AA ONH astrocytes compared to CA ONH astrocytes, obtained by RMA-SAM analysis.(0.11 MB DOC)Click here for additional data file.Table S3Genes differentially expressed in normal AA ONH astrocytes compared to their CA counterpart using Lima. List of genes differentially expressed in AA ONH astrocytes compared to CA ONH astrocytes, obtained by using Limma package in Bioconductor.(0.06 MB DOC)Click here for additional data file.Table S4Functional analysis of genes differentially expressed in AA vs. CA. Functional classification of genes differentially expressed in AA ONH astrocytes compared to and CA ONH astrocytes.(0.06 MB DOC)Click here for additional data file.Table S5Selected Gene ontology for AA-CA comparison. Gene Ontology for genes differentially expressed in AA ONH astrocytes compared to and CA ONH astrocytes.(0.04 MB DOC)Click here for additional data file.Table S6Real-Time PCR validation of microarray expression analysis of normal Caucasian American and African American ONH astrocytes. Real-Time PCR validation of microarray expression analysis of normal CA and AA ONH astrocytes.(0.08 MB DOC)Click here for additional data file.Table S7Quantitative RT-PCR primer information. RT-PCR primer infortion(0.10 MB DOC)Click here for additional data file.Table S8Primary Antibodies Used in this study. Information on the primary antibodies used in this study.(0.04 MB DOC)Click here for additional data file.Text S1Supplemental Methods. Detailed methods for Western Blots, immunohistochemistry and immunocytochemistry.(0.05 MB DOC)Click here for additional data file.


PSA gene. Studies of a single nucleotide polymorphism  in ARE1 of the PSA gene have been conflicting for risk of prostate cancer and effect on plasma PSA levels. In this nested case–control analysis of 500 white cases and 676 age- and smoking-matched white controls in the Physicians' Health Study we evaluated the association of rs266882 with risk and survival of prostate cancer and prediagnostic total and free PSA plasma levels, alone or in combination with AR CAG repeats. We used conditional logistic regression, linear regression and Cox regression, and found no significant associations between rs266882  and overall prostate cancer risk : 0.88–1.67) or prostate cancer-specific survival . Similarly, no associations were found among high grade or advanced stage tumours, or by calendar year of diagnosis. There was no significant association between rs266882 and baseline total or free PSA levels or the AR CAG repeats, nor any interaction associated with prostate cancer risk. Meta-analysis of 12 studies of rs266882 and overall prostate cancer risk was null.Prostate-specific antigen (PSA) is a protease produced in the prostate that cleaves insulin-like growth factor binding protein-3 and other proteins. Production is mediated by the androgen receptor (AR) binding to the androgen response elements (ARE) in the promoter region of the  PSA gene (Prostate-specific antigen (PSA) is a protease produced primarily in the prostate and has widely been used as a diagnostic marker of prostate cancer since the early 1990s (PSA −158 G/A) of the PSA gene (rs266882), identified in 1999  to evaluate the rs266882 polymorphism in prostate cancer. We examined associations with risk, survival and prediagnostic baseline plasma PSA levels, alone and in combination with β-carotene among 22 071 US male physicians, aged 40–84 years in 1982; none had a cancer diagnosis at baseline  on age  and smoking status  to controls selected at random from among men free from diagnosed prostate cancer at the time the case was diagnosed. A total of 676 controls were included. All men provided informed consent, and the protocol was approved by the Institutional Review Board at Partners Healthcare.PSA gene were amplified and the three possible genotypes were distinguished by cutting with the NheI restriction enzyme. The AR gene CAG repeat length was determined by running the PCR-amplified fragments on a denaturing polyacrylamide gel with automated fluorescence detection of the fragments and sizing by Genescan  using conditional logistic regression, matched on age and smoking status, to evaluate the association between the rs266882 polymorphism and prostate cancer risk. We stratified the analysis further according to tumour grade , stage (T1/T2 or T3/T4) and calendar year of diagnosis (1982–1992 or 1992–1995). The following number of cases and matched controls  for each subgroup: low grade , moderate grade , high grade , localized: T1/T2 , or advanced: T3/T4  tumour stage, diagnosed 1982–1992  and diagnosed 1992–1995 .To limit the potential for population stratification, we restricted the analysis to white men (94% of PHS cohort is white). Hardy–Weinberg equilibrium of allelic frequency was tested by a goodness of fit −1, ⩾4 ng ml−1; free PSA: <15%, 15–24%, ⩾25%) or number of AR CAG repeats  using likelihood ratio tests.Additionally, we assessed whether the association of rs266882 genotype and prostate cancer risk differed according to categorical pre-diagnostic PSA levels . We used Cox proportional hazard models to calculate hazard ratios and 95% CI, adjusted for aggressive disease , age at diagnosis and date of diagnosis (pre/post 1992).AR CAG repeats was evaluated to determine if the effect of rs266882 genotype differed according to AR CAG repeat length. The SAS Statistical Software (Version 9.1) was used for these analyses.Linear regression was used to estimate the association among PSA levels  with rs266882 genotype, separately for cases and controls. An interaction with vs AA genotypes and GA vs AA genotypes. Inverse variance weighting with a random effects model was used to create a summary estimate using the statistical package Stata  and 31% were advanced tumour stage at diagnosis (8% missing) . The majP=0.18). Controlling for matching factors, there was no evidence of an association between PSA genotype and total prostate cancer risk. Moreover, we found no association between the rs266882 genotype and cancer risk when we stratified within tumour grade or stage, or among those diagnosed pre/post PSA era  was similar to other studies of white men ( PSA era .P for interaction ∼0.41) or free PSA level (P for interaction ∼0.70).As shown earlier in AR. Shorter AR CAG repeats, which are associated with greater transactivation of the AR, were associated with higher risk of advanced prostate cancer compared with longer AR CAG repeats  and free PSA levels .An earlier study within the PHS cohort examined a trinucleotide CAG repeat polymorphism in Among the 500 men with prostate cancer, 111 men died of their disease. The median survival time between diagnosis and prostate cancer-specific death was 13.5 years (range: <1–24.3 years). We found no association between the rs266882 genotype and prostate cancer survival .PSA −158 G/A) polymorphism (rs266882 genotype) and prostate cancer risk, survival and pre-diagnostic plasma PSA levels. We found no association between the PSA polymorphism and cancer risk, overall or by grade, stage or calendar year of diagnosis. Similarly, the rs266882 genotype was not associated with cancer-specific survival. Finally, there was no association between the rs266882 genotype and PSA plasma levels among cases or controls. There were no significant interactions of the rs266882 genotype with PSA plasma level or AR CAG repeat length for any of the outcomes examined.In this large prospective study, we comprehensively assessed the relation of the ARE1 (β2 (TGF-β2)  reported a positive association of the GG allele of rs266882 with a three-fold risk of advanced cancer  , higher serum PSA levels were statistically significantly associated with AA genotype when all ethnic groups were combined, but not for any of the ethnic groups individually, suggesting population stratification may be a possibility for this finding (−1, men with the AA genotype had a 28% higher level of PSA than men with the GG genotype (−1 (−1 (It has been hypothesised that the rs266882 polymorphism may be associated with serum levels of PSA through higher binding affinity of the ARE1 with either the A or G allele. Results of such studies also have been contradictory. Among healthy controls (PSA levels <4 ng mlAR regulates PSA expression by binding androgen response elements. Some studies of AR, including ours, found shorter CAG repeats associated with increased expression of AR and with higher risk of advanced prostate cancer compared with longer CAG repeats (AR is of interest. PSA gene. Models were adjusted for age and ethnicity, however residual confounding by ethnicity in this very diverse cohort could account for this borderline significant finding. Four additional studies, as well as the present, found no interaction between rs266882 genotype and AR CAG length in relation to plasma PSA levels (The results of this large study with long-term and prospective follow-up, taken together with earlier findings and the results of the meta-analysis, add further support for the conclusion that the rs266882 polymorphism is unrelated to prostate cancer risk, survival or to plasma PSA levels.


Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridization (SSH) or differential display techniques such as cDNA-AFLP (Amplification Fragment Length Polymorphism). Despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed.Crassostrea gigas) to various environmental stressors, we used microarrays and cDNA-AFLP to identify Expressed Sequence Tag (EST) fragments that are differentially expressed in response to bacterial challenge in two heat shock tolerant and two heat shock sensitive full-sib oyster families. We then designed primers for these differentially expressed ESTs in order to validate the results using Q-PCR. For two of these ESTs we tested fourteen primer pairs each and using standard optimization methods , determined that of the fourteen primer pairs tested, six and nine pairs respectively amplified a single product and were thus acceptable for further testing. However, when we used these primers, we obtained different statistical outcomes among primer pairs, raising unexpected but serious questions about their reliability. We hypothesize that as a consequence of high levels of sequence polymorphism in Pacific oysters, Q-PCR amplification is sub-optimal in some individuals because sequence variants in priming sites results in poor primer binding and amplification in some individuals. This issue is similar to the high frequency of null alleles observed for microsatellite markers in Pacific oysters.As part of a larger project aimed at elucidating transcriptome-level responses of Pacific oysters (This study highlights potential difficulties for using Q-PCR as a validation tool for transcriptome analysis in the presence of sequence polymorphism and emphasizes the need for extreme caution and thorough primer testing when assaying genetically diverse biological materials such as Pacific oysters. Our findings suggest that melt-curve analysis alone may not be sufficient as a mean of identifying acceptable Q-PCR primers. Minimally, testing numerous primer pairs seems to be necessary to avoid false conclusions from flawed Q-PCR assays for which sequence variation among individuals produces artifactual and unreliable quantitative results. During the last decade, quantitative real time PCR (Q-PCR) has been widely employed in many fields of biological research  and is considered to be the most sensitive and reliable method of quantifying mRNA transcripts . In contT, the cycle number when sample fluorescence exceeds a chosen threshold above background fluorescence) which is used as an exponent in the estimation procedure, rigorous optimization of Q-PCR assays is especially critical. Even seemingly minor errors and artefacts are greatly magnified by exponentiation.As Q-PCR technology has evolved and its use expanded, diverse protocols using chemistry ranging from non-specific reporter dyes to sequence specific probes and diverse instrumentation have been developed ,5. The sNumerous studies have examined the potential problems and pitfalls of Q-PCR assays ,8,12, hoCrassostrea gigas, a marine bivalve known for its high level of genetic variability  taken from a cDNA-AFLP library , and one EST taken directly from Genbank [GenBank: AJ565694]. We used Q-PCR to quantify the expression levels of these two ESTs. We designed and evaluated 14 primer pairs for each EST sequence and then used 6 and 9 primer pairs respectively that melt curve analysis indicated were suitable for Q-PCR analysis. Results are discussed in light of the impacts of sequence polymorphism on the results of Q-PCR quantification assays.We evaluated the expression of one EST  was initially taken from a previously constructed cDNA-AFLP library. The cDNA used to generate the AFLP profile was reverse-transcribed using SuperScript™ III Reverse Transcriptase and was the result of a normalized pool of cDNA from 16 oysters (full-sib families) challenged with high temperature and bacterial infection . This fragment was cloned into a pCR4-TOPO vector using the TOPO TA Cloning kit (Invitrogen). Sixteen clones were directly sequenced using an ABI 3730XL (Applied Biosystems) automated sequencing system and Big Dye Terminator 3.1 chemistry (Applied Biosystems). The second EST [GenBank: AJ565694] was directly taken from Genbank. The two fragment lengths were respectively 188 and 402 bp. We used Primer Express® Software v2.0 (Applied Biosystems) to design primers for Q-PCR. This software takes into consideration a variety of parameters, including Tm (melting temperature), primer complementarities, and secondary structure, as well as amplicon size. In total, we designed 14 primer pairs for each EST (Table EX956386]).We performed Q-PCR assays targeting two expressed sequence tags (ESTs), both presumed to represent single-copy genes. The first EST [GenBank: ® Green PCR Master Mix (Applied Biosystems) in 25 μl reactions containing cDNA (diluted in 5 μl) and 50 nM  of each primer. Each Q-PCR reaction plate included a non-template negative control to ensure the absence of contamination and the data was normalized using Elongation factor 1 α [GenBank: AB122066] as the reference housekeeping gene. The consistency of Elongation factor 1 α expression was initially evaluated by testing the differences in Ct value within families and triplicates , 95°C for 10 min (AmpliTaq Gold® DNA polymerase activation), 50 cycles of 95°C for 15 s and 60°C for 1 min, and finally, 95°C for 15 min, 60°C for 15 s. The reactions were run and results analyzed using Applied Biosystems 7500 Real Time PCR system (software version 1.4) using the absolute quantification program and included a post-PCR melt curve analysis, to detect nonspecific amplification in cDNA samples. Quantification of gene expression was based on the determination of threshold cycle , defined as the first cycle number with detectable fluorescence above background. The CT value for each sample was estimated using the automatic baseline setting. Relative quantification was accomplished by normalizing raw CT values to the reference gene expressed as target/reference ratios  and [AJ565694] respectively showed a single product in the melt curve analysis and were thus considered to be worth further consideration and testing. All primer pairs that produced multiple products were eliminated from further consideration. Melt curve analyses, raw data, and statistical outcomes are summarized in figures 86 and , it is interesting to observe the similarity of Ct values 28 ± 0.5) across families for primer pair 1 and 3 (table 8 ± 0.5 aTo test this hypothesis, we sequenced 16 clones of the original fragment from the original cDNA-AFLP library. This cDNA is the result of a normalized pool of cDNA collected from 16 individual oysters. An examination of the 16 sequences underlines the presence of polymorphism figure . We obseOverall, primer pairs 1 and 3 seem to be unaffected by the observed polymorphisms while primer pairs 2, 10 and 11 under-estimate the level of expression of Families 4, 25 and 34 relative to Family 65 due to null alleles caused by sequence variation in the priming regions even though all of these primer pairs produce a single product in the melt-curve analysis and are thus acceptable by standard criteria.Turning to our second EST, the same reasoning may be applied, although we do not have access to multiple sequences as for [EX956386]. Pattern A is the most frequent, and is produced by primer pairs 3, 5, 7, 9 and 12. By comparing the Ct values displayed in pattern A (table et al. [There are few examples of how sequence polymorphism affects Q-PCR results in the published literature, but, in a recent study, Stevenson et al.  demonstrOur study demonstrates that careful and rigorous primer optimization and an examination of sequence variation among families or individuals is a critical step before real time PCR assays are used to complement whole transcriptome analyses, especially when dealing with short fragments such as those generated by differential display techniques. Statistical outcomes can be profoundly influenced by polymorphisms in the sequence under study if they cause poor binding of primers or poor amplification. These artefacts cannot be detected using standard melt-curve analyses because they have purely quantitative rather than qualitative effects. For this reason, it is strongly recommended when working with genetically diverse biological material, to test multiple primers and, if at all possible, to examine the sequences investigated for polymorphisms in priming regions to avoid erroneous conclusions.NT participated in study design, carried out the laboratory analyses and drafted the manuscript. RPL participated in the study design and sample collection. MDC participated in study design and was involved in the manuscript preparation. All authors read and approved the final manuscript.


Low toxicity and convenience of administration facilitate use, however clinical resistance is a major limitation. Investigation has failed to fully explain the molecular mechanisms of resistance and no clinically useful predictive biomarkers for 5FU resistance have been identified. We investigated the molecular mechanisms of clinical 5FU resistance in colorectal adenocarcinoma patients in a prospective biomarker discovery project utilising gene expression profiling. The aim was to identify novel 5FU resistance mechanisms and qualify these as candidate biomarkers and therapeutic targets.Putative treatment specific gene expression changes were identified in a transcriptomics study of rectal adenocarcinomas, biopsied and profiled before and after pre-operative short-course radiotherapy or 5FU based chemo-radiotherapy, using microarrays. Tumour from untreated controls at diagnosis and resection identified treatment-independent gene expression changes. Candidate 5FU chemo-resistant genes were identified by comparison of gene expression data sets from these clinical specimens with gene expression signatures from our previous studies of colorectal cancer cell lines, where parental and daughter lines resistant to 5FU were compared. A colorectal adenocarcinoma tissue microarray  was used as an independent set to qualify candidates thus identified.APRIL/TNFSF13 mRNA was significantly upregulated following 5FU based concurrent chemo-radiotherapy and in 5FU resistant colorectal adenocarcinoma cell lines but not in radiotherapy alone treated colorectal adenocarcinomas. Consistent withAPRIL's known function as an autocrine or paracrine secreted molecule, stromal but not tumour cell protein expression by immunohistochemistry was correlated with poor prognosis (p = 0.019) in the independent set. Stratified analysis revealed that protein expression of APRIL in the tumour stroma is associated with survival in adjuvant 5FU treated patients only , and is independently predictive of lack of clinical benefit from adjuvant 5FU .A combined investigative model, analysing the transcriptional response in clinical tumour specimens and cancers cell lines, has identified APRIL, a novel chemo-resistance biomarker with independent predictive impact in 5FU-treated CRC patients, that may represent a target for novel therapeutics. Significant progress has been made recently in the systemic treatment of colorectal adnocarcinoma (CRC). There are currently 8 agents licensed for use in the US and Europe 5-fluorouracil (5FU), floxuridine, capecitabine, irinotecan, oxaliplatin, cetuximab, panitumumab and bevacizumab . CombinaIdentification of the clinically important mechanisms of resistance to 5FU would allow better selection of patients for 5FU therapy and the rationale design of targeted therapeutics to overcome resistance, and thus increase the proportion of patients deriving benefit from 5FU. A predictive biomarker for clinical 5FU resistance would clearly be useful, but progress has been limited in this area and investigation has thus far failed to fully explain the molecular mechanisms that areimportant for clinical 5FU resistance -4. PreclThe global perspective provided by gene expression profiling has provided novel insights into the molecular mechanisms of clinical response to therapy in human cancers , althougThe study was approved by the North of Scotland Research Ethics Committee. Patients provided informed consent in accordance with the regulations and instructions of the North of Scotland Research Ethics Committee for study participation, including use and publication of results. Full clinicopathological details are provided in table Tumour biopsies were collected at the time of endoscopic diagnosis of rectal adenocarcinoma and placed immediately into RNAlater (800 μl) . Tumour biopsies collected at time of curative surgical resection were placed immediately into normal saline and a pathologist provided a representative tumour biopsy, which was placed immediately into RNAlater within 30 minutes (800 μl). Tissues were stored in RNALater at 4°C overnight (16-18 hours), then washed in 500 μl ice cold RNase free PBS  and snap frozen in liquid nitrogen. Long-term storage of tissues was at -80°C. Before RNA extraction, histological diagnosis and features were confirmed by frozen section histology. Extraction and purification of total RNA was performed using TRIZOL reagent  and RNeasy Microkits , according to the manufacturer's instructions. Quantification of total RNA was performed by spectrophometry . Quality of total RNA and cRNA was assessed using a BioAnalyser 2100 . Target preparation for the Affymetrix Genechips™ was according to manufacturer's instructions . Specifically, 4 μg of total RNA was used for reverse transcription and synthesis and amplification of biotin labelled cRNA using the One cycle target labelling and control reagents. Clean-up of biotin-cRNA was performed with RNeasy Minikits . Fragmentation was performed using 20 μg of biotin-labelled cRNA. A hybridisation cocktail was prepared from 15 μg which was first hybridised to Test 3 GeneChips™ to assess sample quality (GAPDH 3':5' < 3 and Actin 3': 5' < 3) and then to HGU133 Plus2.0 GeneChips™ (10 μg) for gene expression analysis. Procedures for hybridisation, washing, staining and scanning of chips were carried out according to standard protocols .Analysis of the gene expression data is described in detail in Additional file Description of the Tissue Microarray (TMA) is provided in previous publications . A total2 test, with Fisher's exact test where appropriate, was used for binary categorical variables, Pearson's χ2 test for non-binary categorical variables and Student's t-test for numerical variables. Kaplan-Meier curves were constructed to assess survival and the log rank test to assess statistical significance. The Cox proportional hazards model was used for multivariate analysis of survival. Two-sided p values of less than 0.05 were considered significant. All analyses were performed using SPSS for Windows, version 13.0 .Continuity corrected χIn a pilot transcriptomics study of rectal cancer patients, we used oligonucleotide microarrays to profile the expression of over 47000 transcripts representing 38562 human genes in rectal tumour biopsies before and after pre-operative treatment with CRT (n = 4 patients); table SOPs were developed and validated to allow collection of tissues at endoscopic diagnosis and at surgical resection, whilst preserving RNA integrity. Total RNA extracted from these tissues (10-30 mg) in this pilot study provided sufficient yield (8 to 40 ug) and quality total RNA for gene expression analysis on Affymetrix oligonucleotide microarrays. Raw gene expression data is provided in MIAME complaint format in Array express, accession number E-MEXP-1901.Additional File Additional file Threshold and probabilistic filtering of the data , irrespective of APRIL protein expression figure . In contGlobal gene expression profiling of clinical response to therapy has provided a useful means for biomarker and novel target discovery in several solid tumours ,13. The Since a small number of rectal adenocarcinomas have been profiled (n = 10), stringent and focussed analysis of the microarray data was applied to identify leads for further investigation. This included hypothesis-driven focus on cell death pathways and comparison with our previously published cell line work. The key candidate was subsequently validated a in larger independent set (n = 234) using a different technique (immunohistochemistry).The biological validity of the experimental model and the data is confirmed by the finding of significant alterations in the gene expression of previously implicated molecules and pathways, for example p21 which has been implicated in numerous studies -25. The A supervised analysis of cell death genes, reveals shared genes and pathways. The analysis supports the hypothesise that initiation of cell death is a common final pathway resulting from a multitude of upstream responses to the insult and resultant cellular stress of cytotoxic chemotherapy or radiotherapy thereby accounting for gene expression overlap seen.The majority of the genes identified in our analysis represent genes and pathways that have not previously been implicated in clinical response of rectal adenocarcinoma or as mechanisms of action or resistance to radiotherapy or 5FU or 5FU-based CRT. This is consistent with the findings of other gene expression profiling studies in rectal adenocarcinoma or other tumour types for radiotherapy or 5FU ,26,28-30APRIL/TNFSF13 was found to be upregulated following CRT but not radiotherapy alone in rectal cancers and was also up-regulated in 5FU resistant cell lines in our previous studies . The bioThis study found that expression of APRIL protein in colorectal tumour stroma was associated with worse survival, but only in those patient's treated with adjuvant 5FU chemotherapy. This relationship was also maintained in a multivariate analysis of 5FU chemotherapy treated Stage III colorectal adenocarcinoma patients , in which the Hazard ratio compares favourably to other previously published putative 5FU predictive biomarkers in colorectal cancer  or positive versus negative tumour cell staining (a), or stratified according to stage Dukes A/Stage I, Dukes B/Stage II and Dukes C/Stage III (c). Figure S7.2. Kaplan-Meier survival plots for APRIL immuno-histochemistry showing that positive staining in the tumour stroma shows no association with survival in Duke's A/Stage I (n = 46) or B/Stage II tumours (n = 86).Click here for file