Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma

BACKGROUND. MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation. RESULTS. We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a) represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b) the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c) the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d) the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC) and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX), VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1, ERBB4, and (SLC12A1, TCF21) respectively. We also found strong anti-correlation between VEGFA and the miR-200 family of microRNA: miR-200a*, 200b, 200c and miR-141. Several identified microRNA/mRNA pairs were validated on an independent set of matched ccRCC/normal samples. The regulation of SEMA6A by miR-141 was verified by a transfection assay. CONCLUSIONS. We describe a simple and reliable method to identify direct gene targets of microRNA in any cancer. The constraints we impose (strong dysregulation signature for microRNA and mRNA levels between tumor/normal samples, evolutionary conservation of seed sequence and strong anti-correlation of expression levels) remove spurious matches and identify a subset of robust, tissue specific, functional mRNA targets of dysregulated microRNA.Cancer Institute of New Jersy; New Jersey Commission for Cacner Research; Lineberger Comprehensive Cancer Center Tissue Procurement and Genomics Core Facility; Crawford Fun

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

BACKGROUND: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. CONCLUSIONS/SIGNIFICANCE: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery

Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma

Abstract Background MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation. Results We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a) represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b) the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c) the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d) the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC) and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX), VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1, ERBB4, and (SLC12A1, TCF21) respectively. We also found strong anti-correlation between VEGFA and the miR-200 family of microRNA: miR-200a*, 200b, 200c and miR-141. Several identified microRNA/mRNA pairs were validated on an independent set of matched ccRCC/normal samples. The regulation of SEMA6A by miR-141 was verified by a transfection assay. Conclusions We describe a simple and reliable method to identify direct gene targets of microRNA in any cancer. The constraints we impose (strong dysregulation signature for microRNA and mRNA levels between tumor/normal samples, evolutionary conservation of seed sequence and strong anti-correlation of expression levels) remove spurious matches and identify a subset of robust, tissue specific, functional mRNA targets of dysregulated microRNA

Clinical course of cone dystrophy caused by mutations in the RPGR gene

Contains fulltext : 97720.pdf (publisher's version ) (Closed access)BACKGROUND: Mutations in the RPGR gene predominantly cause rod photoreceptor disorders with a large variability in clinical course. In this report, we describe two families with mutations in this gene and cone involvement. METHODS: We investigated an X-linked cone dystrophy family (1) with 25 affected males, 25 female carriers, and 21 non-carriers, as well as a small family (2) with one affected and one unaffected male. The RPGR gene was analyzed by direct sequencing. All medical records were evaluated, and all available data on visual acuity, color vision testing, ophthalmoscopy, fundus photography, fundus autofluorescence, Goldmann perimetry, SD-OCT, dark adaptation, and full-field electroretinography (ERG) were registered. Cumulative risks of visual loss were studied with Kaplan-Meier product-limit survival analysis. RESULTS: Both families had a frameshift mutation in ORF15 of the RPGR gene; family 1 had p.Ser1107ValfsX4, and family 2 had p.His1100GlnfsX10. Mean follow up was 13 years (SD 10). Virtually all affected males showed reduced photopic and normal scotopic responses on ERG. Fifty percent of the patients had a visual acuity of <0.5 at age 35 years (SE 2.2), and 75% of the patients was legally blind at age 60 years (SE 2.3). Female carriers showed no signs of ocular involvement. CONCLUSIONS: This report describes the clinical course and visual prognosis in two families with cone dystrophy due to RPGR mutations in the 3' terminal region of ORF15. Remarkable features were the consistent, late-onset phenotype, the severe visual outcome, and the non-expression in female carriers. Expression of RPGR mutations in this particular region appears to be relatively homogeneous and predisposed to cones

Patterns of Evolution and Host Gene Mimicry in Influenza and Other RNA Viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs

Polymorphisms in the RNASE3 Gene Are Associated with Susceptibility to Cerebral Malaria in Ghanaian Children

BACKGROUND: Cerebral malaria (CM) is the most severe outcome of Plasmodium falciparum infection and a major cause of death in children from 2 to 4 years of age. A hospital based study in Ghana showed that P. falciparum induces eosinophilia and found a significantly higher serum level of eosinophil cationic protein (ECP) in CM patients than in uncomplicated malaria (UM) and severe malaria anemia (SA) patients. Single nucleotide polymorphisms (SNPs) have been described in the ECP encoding-gene (RNASE3) of which the c.371G>C polymorphism (rs2073342) results in an arginine to threonine amino acid substitution p.R124T in the polypeptide and abolishes the cytotoxicity of ECP. The present study aimed to investigate the potential association between polymorphisms in RNASE3 and CM. METHODOLOGY/PRINCIPAL FINDINGS: The RNASE3 gene and flanking regions were sequenced in 206 Ghanaian children enrolled in a hospital based malaria study. An association study was carried out to assess the significance of five SNPs in CM (n=45) and SA (n=56) cases, respectively. The two severe case groups (CM and SA) were compared with the non-severe control group comprising children suffering from UM (n=105). The 371G allele was significantly associated with CM (p=0.00945, OR=2.29, 95% CI=1.22-4.32) but not with SA. Linkage disequilibrium analysis demonstrated significant linkage between three SNPs and the haplotype combination 371G/*16G/*94A was strongly associated with susceptibility to CM (p=0.000913, OR=4.14, 95% CI=1.79-9.56), thus, defining a risk haplotype. The RNASE3 371GG genotype was found to be under frequency-dependent selection. CONCLUSIONS/SIGNIFICANCE: The 371G allele of RNASE3 is associated with susceptibility to CM and forms part of a risk associated haplotype GGA defined by the markers: rs2073342 (G-allele), rs2233860 (G-allele) and rs8019343 (A-allele) respectively. Collectively, these results suggest a hitherto unrecognized role for eosinophils in CM pathogenesis

Nutrition, atherosclerosis, arterial imaging, cardiovascular risk stratification, and manifestations in COVID-19 framework: a narrative review.

Background: Atherosclerosis is the primary cause of the cardiovascular disease (CVD). Several risk factors lead to atherosclerosis, and altered nutrition is one among those. Nutrition has been ignored quite often in the process of CVD risk assessment. Altered nutrition along with carotid ultrasound imaging-driven atherosclerotic plaque features can help in understanding and banishing the problems associated with the late diagnosis of CVD. Artificial intelligence (AI) is another promisingly adopted technology for CVD risk assessment and management. Therefore, we hypothesize that the risk of atherosclerotic CVD can be accurately monitored using carotid ultrasound imaging, predicted using AI-based algorithms, and reduced with the help of proper nutrition. Layout: The review presents a pathophysiological link between nutrition and atherosclerosis by gaining a deep insight into the processes involved at each stage of plaque development. After targeting the causes and finding out results by low-cost, user-friendly, ultrasound-based arterial imaging, it is important to (i) stratify the risks and (ii) monitor them by measuring plaque burden and computing risk score as part of the preventive framework. Artificial intelligence (AI)-based strategies are used to provide efficient CVD risk assessments. Finally, the review presents the role of AI for CVD risk assessment during COVID-19. Conclusions: By studying the mechanism of low-density lipoprotein formation, saturated and trans fat, and other dietary components that lead to plaque formation, we demonstrate the use of CVD risk assessment due to nutrition and atherosclerosis disease formation during normal and COVID times. Further, nutrition if included, as a part of the associated risk factors can benefit from atherosclerotic disease progression and its management using AI-based CVD risk assessment

Perceptions of trekking tourism and social and environmental change in Nepal's Himalayas

The Himalayas are among the world’s youngest mountain ranges. In addition to the geologic processes of mountain building and erosion, they are also highly vulnerable to human influenced change, occurring at local, national, regional, and international scales. A photo-elicitation methodology is employed to show how residents perceive those changes from historical perspectives, as well as their current conditions and impacts on their daily lives. Nepal’s Khumbu region has undergone major social and environmental transformations since the 1960s when international trekking first began to influence the area's economy. The current perceptions of Khumbu residents of these changes is assessed through photo-elicitation interviews. Their responses are placed in the historical context of: (i) institutional and political changes, much of which have been driven by national government policies; (ii) social and economic changes, for which the tourism economy has been central; and (iii) environmental changes, reflecting the impacts of resource management and climate change. The mostly positive perceptions of Khumbu residents toward how their region has changed reflects general improvements in the physical and cultural landscapes of the Khumbu over time, as well as its continuing geographic isolation, which has helped to slow the rate of globalization, while also keeping the region a dynamic and popular tourist destination

ErbB2, EphrinB1, Src Kinase and PTPN13 Signaling Complex Regulates MAP Kinase Signaling in Human Cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression

Combinations of Host Biomarkers Predict Mortality among Ugandan Children with Severe Malaria: A Retrospective Case-Control Study

Background: Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings: Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve&gt;0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1-99.9) sensitivity and 88.8% (79.7-94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions: We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria