Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection.

Endothelial and circulating C19MC microRNAs are biomarkers of infantile hemangioma.

Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1-expressing (GLUT-1-expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response.

Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma.

Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

Molecular changes in endometriosis-associated ovarian clear cell carcinoma.

BACKGROUND: Endometriosis is frequently associated with and thought of having propensity to develop into ovarian clear cell carcinoma (OCCC), although the molecular transformation mechanism is not completely understood. METHODS: We employed immunohistochemical (IHC) staining for marker expression along the potential progression continuum. Expression profiling of microdissected endometriotic and OCCC cells from patient-matched formalin-fixed, paraffin-embedded samples was performed to explore the carcinogenic pathways. Function of novel biomarkers was confirmed by knockdown experiments. RESULTS: PTEN was significantly lost in both endometriosis and invasive tumour tissues, while oestrogen receptor (ER) expression was lost in OCCC relative to endometriosis. XRCC5, PTCH2, eEF1A2 and PPP1R14B were significantly overexpressed in OCCC and associated endometriosis, but not in benign endometriosis (p ⩽ 0.004). Knockdown experiments with XRCC5 and PTCH2 in a clear cell cancer cell line resulted in significant growth inhibition. There was also significant silencing of a panel of target genes with histone H3 lysine 27 trimethylation, a signature of polycomb chromatin-remodelling complex in OCCC. IHC confirmed the loss of expression of one such polycomb target gene, the serous ovarian cancer lineage marker Wilms' tumour protein 1 (WT1) in OCCC, while endometriotic tissues showed significant co-expression of WT1 and ER. CONCLUSIONS: Loss of PTEN expression is proposed as an early and permissive event in endometriosis development, while the loss of ER and polycomb-mediated transcriptional reprogramming for pluripotency may play an important role in the ultimate transformation process. Our study provides new evidence to redefine the pathogenic programme for lineage-specific transformation of endometriosis to OCCC.

NAD+ protects against EAE by regulating CD4+ T-cell differentiation.

CD4(+) T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD(+)) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4(+)IFNγ(+)IL-10(+) T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD(+) regulates CD4(+) T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD(+), the frequency of T-bet(-/-) CD4(+)IFNγ(+) T cells was twofold higher than wild-type CD4(+) T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4(+) T-cell differentiation and demonstrate that NAD(+) may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases.

Strategies addressing barriers to clinical trial enrollment of underrepresented populations: a systematic review.

BACKGROUND: Underrepresentation of racial and ethnic minorities in clinical trials remains a reality while they have disproportionately higher rates of health disparities. OBJECTIVE: The purpose of this study was to identify successful community-engaged interventions that included health care providers as a key strategy in addressing barriers to clinical trial enrollment of underrepresented patients. DESIGN: A systematic review of the literature on interventions addressing enrollment barriers to clinical trials for racial and ethnic minorities was performed in Ovid MEDLINE, EBSCO Megafile, and EBSCO CINAHL. The systematic review identified 360 studies, and 20 were selected using the inclusion criteria. An iterative process extracted information from the eligible studies. RESULTS: The 20 selected studies were analyzed and then grouped by first author, nature of the clinical research initiative, priority populations, key strategies, and study outcomes. Nine of the studies addressed cancer clinical trials and 11 related to chronic medical conditions, including diabetes, hypertension management, and chronic kidney disease. The key strategies employed were categorized according to their presumed impact on barriers incurred at distinct steps in study recruitment: clinical trial awareness, opportunity to participate, and acceptance of enrollment. The strategies were further categorized by whether they would address barriers associated with minority perceptions of the research process and barriers related to how studies were designed and implemented. CONCLUSION: Multiple and flexible strategies targeting providers and participants at provider sites and within communities might be needed to enroll underrepresented populations into clinical trials.

MicroRNA-146a and microRNA-155 show tissue-dependent expression in dental pulp, gingival and periodontal ligament fibroblasts in vitro.

MicroRNAs (miRNAs) are small non-coding RNAs showing a tissue-specific expression pattern, and whose function is to suppress protein synthesis. In this study, we hypothesized that expression of miRNAs would differ among fibroblasts from dental pulp (DPF), gingiva (GF) and periodontal ligament (PLF) in vitro. Once established by an explant technique, DPF, GF and PLF were collected for RNA isolation and subjected to a miRNA microarray. Next, cells were stimulated with E. coli lipopolysaccharide (LPS) for 24 h and then collected for RNA isolation. Expression of miR-146a and miR-155 was investigated by qPCR. Microarray screening revealed several miRNAs that showed specifically high expression in at least one of the fibroblast subtypes. These molecules are potentially involved in the regulation of extracellular matrix turnover and production of inflammatory mediators. Microarray analysis showed that both miR-146a and miR-155 were among the miRNAs expressed exclusively in GF. qPCR demonstrated significant upregulation of miR-146a only in GF after LPS stimulation, whereas basal expression of miR-155 was higher in GF than in the other cell subtypes. LPS downregulated the expression of miR-155 only in GF. Our results suggest that the expression and regulation of miR-146a and miR-155 are more pronounced in GF than in DPF and PLF.

Sensory ability in the narwhal tooth organ system.

The erupted tusk of the narwhal exhibits sensory ability. The hypothesized sensory pathway begins with ocean water entering through cementum channels to a network of patent dentinal tubules extending from the dentinocementum junction to the inner pulpal wall. Circumpulpal sensory structures then signal pulpal nerves terminating near the base of the tusk. The maxillary division of the fifth cranial nerve then transmits this sensory information to the brain. This sensory pathway was first described in published results of patent dentinal tubules, and evidence from dissection of tusk nerve connection via the maxillary division of the fifth cranial nerve to the brain. New evidence presented here indicates that the patent dentinal tubules communicate with open channels through a porous cementum from the ocean environment. The ability of pulpal tissue to react to external stimuli is supported by immunohistochemical detection of neuronal markers in the pulp and gene expression of pulpal sensory nerve tissue. Final confirmation of sensory ability is demonstrated by significant changes in heart rate when alternating solutions of high-salt and fresh water are exposed to the external tusk surface. Additional supporting information for function includes new observations of dentinal tubule networks evident in unerupted tusks, female erupted tusks, and vestigial teeth. New findings of sexual foraging divergence documented by stable isotope and fatty acid results add to the discussion of the functional significance of the narwhal tusk. The combined evidence suggests multiple tusk functions may have driven the tooth organ system's evolutionary development and persistence.

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor ß1 (TGF- ß1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Downregulated gene expression of TGF-ßs in diabetic oral wound healing.

BACKGROUND: Healing of tooth extraction sockets in poorly controlled diabetic patients is often delayed and accompanied by severe infection. The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are still not fully understood. OBJECTIVES: The purpose of this study was to investigate molecular changes associated with delayed oral wound healing in diabetes. MATERIALS AND METHODS: Six to eight weeks old male type 2 diabetes and age matched control inbred mice were used and maxillary molar tooth extractions were performed. At 4 and 7 days after tooth extraction, the edentulous mucosa of the mice were harvested, and analyzed for histology and gene expression of key wound healing factors. RESULTS: In the diabetic model, histological analysis showed that epithelial tissue migration for wound closure was delayed after tooth extraction compared to the control. Quantitative real-time PCR revealed that expression of the TGF-ß1, TGF-ß2, TGF-ß3, TGFßRII and TGFßRIII genes was significantly downregulated in the diabetic model at 4 and 7 days after tooth extraction. CONCLUSION: These results suggest that delayed wound healing of oral mucosa in diabetes may be associated with decreased expression levels of these regulatory genes which play important roles in controlling epithelial wound closure.

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling.

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.

Performance comparison of multiple microarray platforms for gene expression profiling.

With genome-wide gene expression microarrays being increasingly applied in various areas of biomedical research, the diversity of platforms and analytical methods has made comparison of data from multiple platforms very challenging. In this chapter, we describe a generalized framework for systematic comparisons across gene expression profiling platforms, which could accommodate both the available commercial arrays and "in-house" platforms, with both one-dye and two-dye platforms. It includes experimental design, data preprocessing protocols, cross-platform gene matching approaches, measures of data consistency comparisons, and considerations in biological validation. In the design of this framework, we considered the variety of platforms available, the need for uniform quality control procedures, real-world practical limitations, statistical validity, and the need for flexibility and extensibility of the framework. Using this framework, we studied ten diverse microarray platforms, and we conclude that using probe sequences matched at the exon level is important to improve cross-platform data consistency compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values, as confirmed by QRT-PCR. After stringent preprocessing, commercial arrays were more consistent than "in-house" arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.

Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells.

BACKGROUND: The small molecule Halofuginone (HF) is a potent regulator of extracellular matrix (ECM ) gene expression and is unique in its therapeutic potential. While the basis for HF effects is unknown, inhibition of TGFß signaling and activation of the amino acid restriction response (AAR) have been linked to HF transcriptional control of a number of ECM components and amelioration of fibrosis and alleviation of autoimmune disease by regulation of Th17 cell differentiation, respectively. The aim of this study was to generate a global expression profile of HF targets in epithelial cells to identify potential mediators of HF function in this cell type. RESULTS: We report that HF modulation of the expression of the ECM remodeling protein Mmp13 in epithelial cells is separable from previously reported effects of HF on TGFß signal inhibition, and use microarray expression analysis to correlate this with transcriptional responses characteristic of the Integrated Stress Response (ISR). CONCLUSIONS: Our findings suggest activation of the ISR may be a common mechanism underlying HF biological effects.

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.

2-DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2-D gels. Of the 588 matched proteins separated on 2-D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein-specific staining was corroborated by the identification of 16 phosphoproteins by nano-LC MS/MS and phosphotyrosine kinase activity assay.

Two developmental modules establish 3D beak-shape variation in Darwin's finches.

Bird beaks display tremendous variation in shape and size, which is closely associated with the exploitation of multiple ecological niches and likely played a key role in the diversification of thousands of avian species. Previous studies have demonstrated some of the molecular mechanisms that regulate morphogenesis of the prenasal cartilage, which forms the initial beak skeleton. However, much of the beak diversity in birds depends on variation in the premaxillary bone. It forms later in development and becomes the most prominent functional and structural component of the adult upper beak/jaw, yet its regulation is unknown. Here, we studied a group of Darwin's finch species with different beak shapes. We found that TGFßIIr, ß-catenin, and Dickkopf-3, the top candidate genes from a cDNA microarray screen, are differentially expressed in the developing premaxillary bone of embryos of species with different beak shapes. Furthermore, our functional experiments demonstrate that these molecules form a regulatory network governing the morphology of the premaxillary bone, which differs from the network controlling the prenasal cartilage, but has the same species-specific domains of expression. These results offer potential mechanisms that may explain how the tightly coupled depth and width dimensions can evolve independently. The two-module program of development involving independent regulating molecules offers unique insights into how different developmental pathways may be modified and combined to induce multidimensional shifts in beak morphology. Similar modularity in development may characterize complex traits in other organisms to a greater extent than is currently appreciated.

AnyExpress: integrated toolkit for analysis of cross-platform gene expression data using a fast interval matching algorithm.

BACKGROUND: Cross-platform analysis of gene express data requires multiple, intricate processes at different layers with various platforms. However, existing tools handle only a single platform and are not flexible enough to support custom changes, which arise from the new statistical methods, updated versions of reference data, and better platforms released every month or year. Current tools are so tightly coupled with reference information, such as reference genome, transcriptome database, and SNP, which are often erroneous or outdated, that the output results are incorrect and misleading. RESULTS: We developed AnyExpress, a software package that combines cross-platform gene expression data using a fast interval-matching algorithm. Supported platforms include next-generation-sequencing technology, microarray, SAGE, MPSS, and more. Users can define custom target transcriptome database references for probe/read mapping in any species, as well as criteria to remove undesirable probes/reads. AnyExpress offers scalable processing features such as binding, normalization, and summarization that are not present in existing software tools. As a case study, we applied AnyExpress to published Affymetrix microarray and Illumina NGS RNA-Seq data from human kidney and liver. The mean of within-platform correlation coefficient was 0.98 for within-platform samples in kidney and liver, respectively. The mean of cross-platform correlation coefficients was 0.73. These results confirmed those of the original and secondary studies. Applying filtering produced higher agreement between microarray and NGS, according to an agreement index calculated from differentially expressed genes. CONCLUSION: AnyExpress can combine cross-platform gene expression data, process data from both open- and closed-platforms, select a custom target reference, filter out undesirable probes or reads based on custom-defined biological features, and perform quantile-normalization with a large number of microarray samples. AnyExpress is fast, comprehensive, flexible, and freely available at http://anyexpress.sourceforge.net.

A potential role for intragenic miRNAs on their hosts' interactome.

BACKGROUND: miRNAs are small, non-coding RNA molecules that mainly act as negative regulators of target gene messages. Due to their regulatory functions, they have lately been implicated in several diseases, including malignancies. Roughly half of known miRNA genes are located within previously annotated protein-coding regions ("intragenic miRNAs"). Although a role of intragenic miRNAs as negative feedback regulators has been speculated, to the best of our knowledge there have been no conclusive large-scale studies investigating the relationship between intragenic miRNAs and host genes and their pathways. RESULTS: miRNA-containing host genes were three times longer, contained more introns and had longer 5' introns compared to a randomly sampled gene cohort. These results are consistent with the observation that more than 60% of intronic miRNAs are found within the first five 5' introns. Host gene 3'-untranslated regions (3'-UTRs) were 40% longer and contained significantly more adenylate/uridylate-rich elements (AREs) compared to a randomly sampled gene cohort. Coincidentally, recent literature suggests that several components of the miRNA biogenesis pathway are required for the rapid decay of mRNAs containing AREs. A high-confidence set of predicted mRNA targets of intragenic miRNAs also shared many of these features with the host genes. Approximately 20% of intragenic miRNAs were predicted to target their host mRNA transcript. Further, KEGG pathway analysis demonstrated that 22 of the 74 pathways in which host genes were associated showed significant overrepresentation of proteins encoded by the mRNA targets of associated intragenic miRNAs. CONCLUSIONS: Our findings suggest that both host genes and intragenic miRNA targets may potentially be subject to multiple layers of regulation. Tight regulatory control of these genes is likely critical for cellular homeostasis and absence of disease. To this end, we examined the potential for negative feedback loops between intragenic miRNAs, host genes, and miRNA target genes. We describe, how higher-order miRNA feedback on hosts' interactomes may at least in part explain correlation patterns observed between expression of host genes and intragenic miRNA targets in healthy and tumor tissue.

Effects of nicotine on gene expression and osseointegration in rats.

BACKGROUND: While many studies have focused on the hazardous effects of smoking, there is little direct evidence regarding the specific detrimental effects of the nicotine on the osseointegration of implants. OBJECTIVE: To understand the effects of nicotine on gene expression and osseointegration of titanium implants in rats. MATERIAL AND METHODS: Forty-four rats were administered with nicotine or saline for a period of 8 weeks. The femurs were then harvested and analyzed using a three-point bending test. Osseointegration level was determined using bone/implant contact ratio at 2 or 4 weeks after implants were placed. Expression levels of bone matrix-related genes were measured by quantitative real-time polymerase chain reaction. RESULTS: The results of the three-point bending showed that there was no significant difference detected in stiffness between control and nicotine groups at 8 weeks post-saline/nicotine delivery (P=0.705). The bone/implant contact ratio in nicotine-delivered group was significantly decreased compared with those in the control group at 4 weeks (P<0.05). Also, expression levels of osteopontin, type II collagen, bone morphogenic protein-2, bone sialoprotein, and core-binding factor α-1 were significantly down-regulated in the nicotine-delivered group compared with the control. CONCLUSIONS: Although systemic exposure to nicotine did not affect rat bone development, bone wound healing around the implant after placement was affected. These findings suggest that nicotine might inhibit the bone matrix-related gene expressions required for wound healing and thereby diminish implant osseointegration at late stage.

Error margin analysis for feature gene extraction.

BACKGROUND: Feature gene extraction is a fundamental issue in microarray-based biomarker discovery. It is normally treated as an optimization problem of finding the best predictive feature genes that can effectively and stably discriminate distinct types of disease conditions, e.g. tumors and normals. Since gene microarray data normally involves thousands of genes at, tens or hundreds of samples, the gene extraction process may fall into local optimums if the gene set is optimized according to the maximization of classification accuracy of the classifier built from it. RESULTS: In this paper, we propose a novel gene extraction method of error margin analysis to optimize the feature genes. The proposed algorithm has been tested upon one synthetic dataset and two real microarray datasets. Meanwhile, it has been compared with five existing gene extraction algorithms on each dataset. On the synthetic dataset, the results show that the feature set extracted by our algorithm is the closest to the actual gene set. For the two real datasets, our algorithm is superior in terms of balancing the size and the validation accuracy of the resultant gene set when comparing to other algorithms. CONCLUSION: Because of its distinct features, error margin analysis method can stably extract the relevant feature genes from microarray data for high-performance classification.