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3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals. However, most solid tumors are composed of multiple cell types, including fibroblasts. In this study we developed multicellular tumor heterospheroids composed of cancer and fibroblasts cell lines. We developed heterospheroids by combining HT-29, MCF-7, PANC-1 or SW480 with 1BR.3.G fibroblasts, which we have previously reported support spheroid formation. We also tested fibroblast cell lines, MRC-5, GM00498 and HIF, but 1BR.3.G was found to best form heterospheroids with morphological similarity to in vivo tumor tissue. The architectural organization of heterospheroids was based on histological examination using immunohistochemistry. We found that HT-29 and MCF-7 cells developed spheroids with the cancer cells surrounding the fibroblasts, whereas PANC-1 cells interspersed with the fibroblasts and SW480 cells were surrounded by fibroblasts. The fibroblasts also expressed collagen-1 and FAP-α, and whole transcriptomic analysis (WTA) showed abundant ECM- and EMT-related expression in heterospheroids, thus reflecting a representative tumor-like microenvironment. The WTA showed that PANC-1 heterospheroids possess a strong EMT profile with abundant Vimentin and CDH2 expression. Drug testing was evaluated by measuring cytotoxicity of 5FU and cisplatin using cell viability and apoptosis assays. We found no major impact on the cytotoxicity when fibroblasts were added to the spheroids. We conclude that the cancer cell lines together with fibroblasts shape the architectural organization of heterospheroids to form tumor-like morphology, and we propose that the various 3D tumor structures can be used for drug testing directed against the cancer cells as well as the fibroblasts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nielsen, Madsen, Larsen, Skandorff, Gad and Holmstrøm.)

Differential diagnosis of inflammatory bowel disease (IBD) to Crohn's disease (CD) or ulcerative colitis (UC) is crucial for treatment decision making. With the aim of generating a clinically applicable molecular-based tool to classify IBD patients, we assessed whole transcriptome analysis on endoscopy samples. A total of 408 patient samples were included covering both internal and external samples cohorts. Whole transcriptome analysis was performed on an internal cohort of FFPE IBD samples (CD, n = 16 and UC, n = 17). The 100 most significantly differentially expressed genes (DEG) were tested in two external cohorts. Ten of the DEG were further processed by functional enrichment analysis from which seven were found to show consistent significant performance in discriminating CD from UC: PI3, ANXA1, VDR, MTCL1, SH3PXD2A-AS1, CLCF1, and CD180. Differential expression of PI3, ANXA1, and VDR was reproduced by RT-qPCR, which was performed on an independent sample cohort of 97 patient samples (CD, n = 44 and UC, n = 53). Gene expression levels of the three-gene profile, resulted in an area under the curve of 0.84 (P = 0.02) in discriminating CD from UC, and therefore appear as an attractive molecular-based diagnostic tool for clinicians to distinguish CD from UC., (© 2023. The Author(s).)

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus , using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847-treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus . Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound., Competing Interests: AP was employed by company Unibrains. LS and KH were employed by company Bioneer A/S. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ronco, Kappel, Aragao, Biagi, Svenningsen, Christensen, Permin, Saaby, Holmstrøm, Klitgaard, Sabat, Akkerboom, Monaco, Tinelli, Friedrich, Jana and Olsen.)

Cancer cell spheroids are considered important preclinical tools to evaluate the efficacy of new drugs. In cancer cell spheroids, the cells assemble and grow in 3D structures with cell contact interactions that are partly impermeable, which leads to central hypoxia and necrosis. The cell spheroids thus possess several features identified in clinical tumors. Not only will the effect and behavior of therapeutic drugs in 3D cell spheroids be affected more similarly than in cells grown on culture plates, but molecular interactions and signaling pathways in cells are also more likely to mimic the in vivo situation. The monitoring of various biomarkers including lncRNAs in 3D cell spheroids is important to assess a potentially induced phenotype in the cells and the effects of drugs. Specifically, for lncRNAs, in situ localization can be done using locked nucleic acid (LNA) probe technology. Here we present a protocol for preparation of cell spheroids for use in LNA probe-based in situ hybridization to study lncRNA expression in paraffin embedded 3D cancer cell spheroids.

Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing., Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers., Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio., Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images., Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

Objective: Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied., Approach and Results: Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457)., Conclusions: Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Danielle van Keulen and Dennie Tempel are employed by Quorics B.V., Maarten D Sollewijn Gelpke by Molecular Profiling Consulting, Lori L Jennings by Novartis and Kim Holmstrøm and Boye Schnack Nielsen PhD by Bioneer A/S. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials,as detailed online in the guide for authors.

Some lactic acid bacteria, especially Lactobacillus spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic Lactobacillus plantarum strains, WCSF1 and 299v, display highly different mannose-specific adhesion, with L. plantarum 299v being superior to L. plantarum WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the msa gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger msa transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the msa gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the msa transcript. In addition, the ribosome binding site upstream of the msa gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the msa transcript in the variants than in the wild type, correlating with a strong-Msa phenotype. IMPORTANCE Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In Lactobacillus plantarum , interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the msa gene in L. plantarum strain WCFS1. Strains with increased msa expression could be valuable in the development and manufacture of improved probiotic products., (Copyright © 2019 American Society for Microbiology.)

MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples ( n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.

MicroRNAs are short (18-23 nucleotides) noncoding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA-expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter, we present a fluorescence-based detection method that allows the combination of microRNA in situ hybridization with immunohistochemical staining of one and, in this updated version of the paper, two protein markers detected with primary antibodies raised in the same host species.

Variants in the PLPP3 gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1-6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing in situ mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. In vitro , PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.

Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: microRNAs (miRNAs) are small noncoding RNAs that guide degradation of mRNA and regulate protein expression. miRNA based diagnostic biomarkers for ulcerative colitis (UC) and Crohn's disease (CD) are emerging but information about the cellular localization of many miRNAs is limited and more detailed histologic evaluation of miRNA expression patterns is needed to understand their immunobiological function., Methods: Formalin-fixed paraffin-embedded colon biopsies from 10 patients with UC and 8 patients with CD together with 9 controls were examined by RT-qPCR and quantitative in situ hybridization (ISH). The cellular expression of miR-21 positive cells was further characterized using immunohistochemical cellular markers., Results: Increased levels of miR-21 and miR-126 were found in UC compared with controls and increased levels of miR-21 were observed in UC compared with CD by both RT-qPCR and quantitative in situ hybridization. miR-126 was localized to endothelial cells and miR-21 to cells in the lamina propria. Multiplex immunohistochemical staining showed miR-21 expression in subsets of CD68 macrophages and CD3 T cells in UC, however, far the majority of the miR-21 positive cells could not be categorized among CD68, CD3, and CD19 cells., Conclusions: This study shows that miR-126 levels are increased in UC and expressed in endothelial cells. miR-21 is expressed in subsets of monocytes/macrophages and T cells and may work as a potential biomarker to distinguish UC from CD. Quantitative in situ hybridization may be a powerful tool for such analysis as it combines overall expression with validation of cellular origin. Studies in larger cohorts may confirm this for clinical diagnostics.

TL1A is a TNF-like cytokine which has been shown to co-stimulate TH1 and TH17 responses during chronic inflammation. The expression of this novel cytokine has been investigated in inflammatory disorders like rheumatoid arthritis and inflammatory bowel disease, but little is known about expression and induction in psoriasis. Indeed, the pathogenesis in psoriasis is still not fully understood and it is speculated that cytokines other than TNF-α are important in subsets of patients. Also, for patients with severe disease that are treated with systemic anti-TNF-α blockade, novel candidates to be used as disease and response biomarkers are of high interest. Here, we demonstrate TL1A expression in biopsies from psoriatic lesions. Also, we investigated spontaneous and induced TL1A secretion from PBMCs and blood levels from a cohort of psoriasis patients. Here, increased spontaneous secretion from PBMCs was observed as compared to healthy controls and a small subset of patients had highly elevated TL1A in the blood. Interestingly, activation of PBMCs with various cytokines showed a decreased sensitivity for TL1A activation in psoriasis patients compared to healthy controls.TL1A levels in blood and biopsies could not be correlated with disease activity with this patient cohort. Thus, additional large-scale studies are warranted to investigate TL1A as a biomarker., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

CD4 + CD25+ regulatory T cells (Tregs) are believed to be pivotal in controlling chronic inflammation as well as in opposing the effect of cancer immunotherapy. Therefore, identification of novel drug compounds that interfere with Treg function is of high priority together with research that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell sorting (FACS) sorted CD4 + CD25(high)CD127(dim/-)CD45RA+ naïve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use of highly pure Tregs in a functional assay based on Treg mediated inhibition of effector T cell proliferation. We report highly reproducible Treg function in assays that test the effect of well-known model compounds such as CpG-A, anti-IL-6R (tocilizumab), anti-TNF-α (adalimumab) or a combination of IL-6 and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology.

Aims/hypothesis: We aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins., Methods: We measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats., Results: We identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro., Conclusions: Type 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute a new group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into three major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells, or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients who were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings show that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab.

Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified miRNA-34a (miR-34a) and its target protein networks as modulator of osteoblastic (OB) differentiation of hMSC. miRNA array profiling and further validation by quantitative RT-PCR revealed that miR-34a was upregulated during OB differentiation of hMSC, and in situ hybridization confirmed its OB expression in vivo. Overexpression of miR-34a inhibited early commitment and late OB differentiation of hMSC in vitro, whereas inhibition of miR-34a by anti-miR-34a enhanced these processes. Target prediction analysis and experimental validation confirmed Jagged1 (JAG1), a ligand for Notch 1, as a bona fide target of miR-34a. siRNA-mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25 homolog A were among miR-34a targets. Furthermore, in a preclinical model of in vivo bone formation, overexpression of miR-34a in hMSC reduced heterotopic bone formation by 60%, and conversely, in vivo bone formation was increased by 200% in miR-34a-deficient hMSC. miRNA-34a exhibited unique dual regulatory effects controlling both hMSC proliferation and OB differentiation. Tissue-specific inhibition of miR-34a might be a potential novel therapeutic strategy for enhancing in vivo bone formation., (© AlphaMed Press.)

In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.

Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.

A microfluidic component library for building systems driving parallel or serial microfluidic-based assays is presented. The components are a miniaturized eight-channel peristaltic pump, an eight-channel valve, sample-to-waste liquid management, and interconnections. The library of components was tested by constructing various systems supporting perfusion cell culture, automated DNA hybridizations, and in situ hybridizations. The results showed that the MainSTREAM components provided (1) a rapid, robust, and simple method to establish numerous fluidic inputs and outputs to various types of reaction chips; (2) highly parallel pumping and routing/valving capability; (3) methods to interface pumps and chip-to-liquid management systems; (4) means to construct a portable system; (5) reconfigurability/flexibility in system design; (6) means to interface to microscopes; and (7) compatibility with tested biological methods. It was found that LEGO Mindstorms motors, controllers, and software were robust, inexpensive, and an accessible choice as compared with corresponding custom-made actuators. MainSTREAM systems could operate continuously for weeks without leaks, contamination, or system failures. In conclusion, the MainSTREAM components described here meet many of the demands on components for constructing and using microfluidics systems.

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human transforming growth factor beta 1, and vascular endothelial growth factor extracted from the patch were determined as 127 ng (±95% CI 20), 92 ng (±95%CI 17), and 1.35 ng (±95%CI 0.37), respectively. We showed a continued release of PDGF-AB over several days, the rate of which was increased by the addition of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important for acute wound healing, could be grown from the patch. The relevance of these findings in relation to the use of the patch for treating recalcitrant wounds is discussed., (© 2012 by the Wound Healing Society.)

microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

MicroRNAs are short (18-23 nucleotides) non-coding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter we present a fluorescence-based technology that allows the combination of microRNA in situ hybridization with immunohistochemistry exemplified by the in situ detection of miR-21 and miR-205 in combination with PDCD4 and smooth muscle a-actin.

A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in combination or sequentially through the ability of the HistoFlex to monitor assays without disassembly.

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low-copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA-based probes and the optimized ISH assay enable simple and fast detection of low-copy number miRNA targets, such as miR-130a in mouse brain.

Dendritic cells (DCs) exposed to tumor antigens followed by treatment with T(h)1-polarizing differentiation signals have paved the way for the development of DC-based cancer vaccines. Critical parameters for assessment of the optimal functional state of DCs and prediction of the vaccine potency of activated DCs have in the past been based on measurements of differentiation surface markers like HLA-DR, CD80, CD83, CD86, and CCR7 and the level of secreted cytokines like interleukin-12p70. However, the level of these markers does not provide a complete picture of the DC phenotype and may be insufficient for prediction of clinical outcome for DC-based therapy. We therefore looked for additional biomarkers by investigating the differential expression of microRNAs (miRNAs) in mature DCs relative to immature DCs. A microarray-based screening revealed that 12 miRNAs were differentially expressed in the two DC phenotypes. Of these, four miRNAs, hsa-miR-155, hsa-miR-146a, hsa-miR-125a-5p, and hsa-miR-29a, were validated by real-time polymerase chain reaction and northern blotting. The matured DCs from 12 individual donors were divided into two groups of highly and less differentiated DCs, respectively. A pronounced difference at the level of miRNA induction between these two groups was observed, suggesting that quantitative evaluation of selected miRNAs potentially can predict the immunogenicity of DC vaccines.

The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.

Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.

Although carbon dioxide (CO(2)) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO(2) concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N(2), and 100% CO(2). The CO(2)-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO(2) as to other anaerobic, slightly acidic environments (100% N(2), pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO(2) and N(2) at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO(2)-containing atmospheres. Together, our findings demonstrate that the CO(2)-response is a partly strain-dependent complex mechanism. The possible links between the CO(2)-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (=CCUG 47106(T)=DSM 15176(T)).

It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes, pbp2229, hpk1021, and lmo2487, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively. In the present work, we determined the direct role of the three genes in nisin resistance. Interruption of pbp2229 and hpk1021 eliminated the nisin resistance phenotype. Interruption of hpk1021 additionally abolished the increase in pbp2229 expression. The results indicate that this nisin resistance mechanism is caused directly by the increase in pbp2229 expression, which in turn is brought about by the increase in hpk1021 expression. We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms. The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (prfA-regulated as well as prfA-independent genes). Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development.

The antagonistic interaction between a potential fish probiont, Pseudomonas fluorescens strain AH2, and its target organism, Vibrio anguillarum, was investigated by studying the genetic response of the target organism when it was exposed to the antagonist. We compared the differential display of arbitrarily PCR-amplified gene transcripts in V. anguillarum serotype O1 when it was exposed to AH2 supernatant with the display of transcripts in nonexposed control cultures. Growth of V. anguillarum was immediately arrested when the organism was exposed to 50% (vol/vol) AH2 supernatant. A total of 10 potentially differentially expressed transcripts were identified. Among these we identified a gene homologous to rpoS that was induced in a dose-dependent manner when V. anguillarum was cultured in media supplemented with sterile filtered supernatant from AH2. rpoS was also induced when growth was arrested with the iron chelator 2,2-dipyridyl. A chromosomal transcript homologous to vibE that participates in vibriobactin synthesis in Vibrio cholerae was also upregulated during AH2 exposure. This transcript could represent a functionally active gene in V. anguillarum involved in biosynthesis of anguibactin or another V. anguillarum siderophore. On the pJM1 plasmid of V. anguillarum serotype O1, a pseudogene designated open reading frame E (ORF E) that contains a frameshift mutation was previously identified. The gene homologous to vibE identified in this study, interestingly, also has significant homology to ORF E on the amino acid level and does not possess the frameshift mutation. Thus, the chromosomally encoded vibE homologue could fulfil the role of the inactive plasmid-encoded ORF E pseudogene. Addition of Fe(3+) to the system eliminated the growth arrest, and the genes homologous to rpoS and vibE were not induced. To our knowledge, this is the first study linking rpoS induction to iron starvation. Taken together, the results of this study suggest that a major part of the antagonistic property exhibited by strain AH2 is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen V. anguillarum and complete growth arrest.