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Abstract Background Physical molecular interactions are the basis of intracellular signalling and gene regulatory networks, and comprehensive, accessible databases are needed for their discovery. Highly correlated transcripts may reflect important functional associations, but identification of such associations from primary data are cumbersome. We have constructed and adapted a user-friendly web application to discover and identify putative macromolecular associations in human peripheral blood based on significant correlations at the transcriptional level. Methods The blood transcriptome was characterized by quantification of 17,328 RNA species, including 341 mature microRNAs in 105 clinically well-characterized postmenopausal women. Intercorrelation of detected transcripts signal levels generated a matrix with > 150 million correlations recognizing the human blood RNA interactome. The correlations with calculated adjusted p-values were made easily accessible by a novel web application. Results We found that significant transcript correlations within the giant matrix reflect experimentally documented interactions involving select ubiquitous blood relevant transcription factors (CREB1, GATA1, and the glucocorticoid receptor (GR, NR3C1)). Their responsive genes recapitulated up to 91% of these as significant correlations, and were replicated in an independent cohort of 1204 individual blood samples from the Framingham Heart Study. Furthermore, experimentally documented mRNAs/miRNA associations were also reproduced in the matrix, and their predicted functional co-expression described. The blood transcript web application is available at http://app.uio.no/med/klinmed/correlation-browser/blood/index.php and works on all commonly used internet browsers. Conclusions Using in silico analyses and a novel web application, we found that correlated blood transcripts across 105 postmenopausal women reflected experimentally proven molecular associations. Furthermore, the associations were reproduced in a much larger and more heterogeneous cohort and should therefore be generally representative. The web application lends itself to be a useful hypothesis generating tool for identification of regulatory mechanisms in complex biological data sets.

Abstract Background To identify candidate tear fluid biomarkers in patients with unilateral acute anterior uveitis (AAU) that can aid in the differentiation between these patients and patients with bacterial keratitis or healthy controls. Methods Thirteen patients (40.1 ± 16.2 years of age) with unilateral AAU, seven patients with unilateral bacterial keratitis (40.2 ± 15.3 years of age), and 14 healthy subjects (41.1 ± 11.6 years of age) were included. The tear proteome of affected eyes was compared with that of the unaffected eye or healthy controls. Proteins were identified by liquid chromatography tandem mass spectrometry and enzyme-linked immunosorbent assay. Results Relative protein ratios were detected and calculated for 272 unique proteins. Compared with healthy controls and the unaffected eye, the top upregulated proteins in AAU eyes were submaxillary gland androgen regulated protein 3B (SMR3B) and SMR3A. Similarly, the top upregulated proteins in bacterial keratitis were S100 calcium-binding protein A9 and orosomucoid 2. The acute phase response protein Serpin Family A Member 3 (SERPINA3) was increased in the healthy eye of AAU patients (P = 0.019) compared with healthy controls. Laser flare measurements in affected eyes of AAU patients showed positive logarithmic correlation with SERPINA3 in tear samples of the unaffected eye (P = 0.022). The use of SERPINA3 as a tear biomarker yielded a sensitivity of 85% and a specificity of 71% in detecting patients with AAU in the study population. Conclusions The acute phase response protein SERPINA3 was increased in tear samples of unaffected eyes of patients with unilateral AAU compared with healthy controls. This study highlights SERPINA3 as a potential biomarker for AAU. Future research should explore the dynamic properties of SERPINA3 in the tear fluid of active and quiescent uveitis eyes.

Abstract Purpose To determine whether unilateral acute anterior uveitis (AAU) induces ipsilateral changes in the tear fluid proteome. Methods Five patients (25–77 years old) with unilateral AAU were included. Tear fluid samples were obtained using Schirmer’s test strips. The healthy eye served as control. Proteins were identified by liquid chromatography tandem mass spectrometry. Results Two hundred forty-two tear fluid sample proteins were identified, of which 75 were present in at least three patients. Nine proteins were at least 1.5-fold increased, whereas eight were at least 1.5-fold decreased in tears from the diseased eye compared with the healthy eye. APOBEC3A was significantly increased (1.43-fold; P = 0.04), whereas TGM2 was significantly decreased (− 1.21-fold; P = 0.03) in tears from the diseased eye relative to the healthy eye. Ingenuity Pathway Analysis identified LXR/RXR (P < 1.02E−4) as a top canonical pathway. Conclusion Unilateral AAU induced detectable changes in the ipsilateral tear fluid proteome and involvement of the inflammation-associated LXR/RXR pathway.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism—MEPE, SOST, WIF1, and DKK1—showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.

PURPOSE:Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. MATERIALS AND METHODS:Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. RESULTS:Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. CONCLUSION:HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.

BACKGROUND: Molecular mechanisms explaining age-related changes in the bone marrow with reduced precursor B cell output are poorly understood. METHODS: We studied the transcriptome of five precursor B cell subsets in individual bone marrow samples from 4 healthy children and 4 adults employing GeneChip® Human Exon 1.0 ST Arrays (Affymetrix®) and TaqMan® Array MicroRNA Cards (Life Technologies™). RESULTS: A total of 1796 mRNAs (11%) were at least once differentially expressed between the various precursor B cell subsets in either age group (FDR 0.1%, p≤1.13×10(-4)) with more marked cell stage specific differences than those related to age. In contrast, microRNA profiles of the various precursor B cell subsets showed less hierarchical clustering as compared to the corresponding mRNA profiles. However, 17 of the 667 microRNA assays (2.5%) were at least once differentially expressed between the subsets (FDR 10%, p≤0.004). From target analysis (Ingenuity® Systems), functional assignment between postulated interacting mRNAs and microRNAs showed especially association to cellular growth, proliferation and cell cycle regulation. One functional network connected up-regulation of the differentiation inhibitor ID2 mRNA to down-regulation of the hematopoiesis- or cell cycle regulating miR-125b-5p, miR-181a-5p, miR-196a-5p, miR-24-3p and miR-320d in adult PreBII large cells. Noteworthy was also the stage-dependent expression of the growth promoting miR-17-92 cluster, showing a partly inverse trend with age, reaching statistical significance at the PreBII small stage (up 3.1-12.9 fold in children, p = 0.0084-0.0270). CONCLUSIONS: The global mRNA profile is characteristic for each precursor B cell developmental stage and largely similar in children and adults. The microRNA profile is much cell stage specific and not changing much with age. Importantly, however, specific age-dependent differences involving key networks like differentiation and cellular growth may indicate biological divergence and possibly also altered production potential with age.

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.

BACKGROUND: The skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored. METHODOLOGY: To address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina. PRINCIPAL FINDINGS: In the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling. CONCLUSIONS AND SIGNIFICANCE: This study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified.