Abstract Prostate cancer (PC) is one of the most common cancers in men worldwide, but current risk-stratification biomarkers are limited in sensitivity and specificity. PC mortality in Black men is twice that of White men in Michigan, and in Detroit, three times greater. Recent assessments of small non-coding regulatory RNAs, including microRNAs (miR) and tRNA fragments (tRFs), as potential low-cost, non-invasive screening biomarkers have shown expression profiles that may predict PC aggressiveness across race. Thus, we conducted a pilot study evaluating miR and tRF as PC risk stratification biomarkers among self-reported African American/Black (AA) and White men. RNA from patient tissue (MRI-identified target and non-target biopsy) was extracted, sequenced, and assessed for mature human miRs and tRFs via miRbase and oncotRF. Using p-value-corrected univariate negative binomial regression, we assessed differential expression of miR and tRF across race, age at biopsy, aggressiveness (Gleason grade group 1, 2-3, 4-5), biopsy site, and cancer status (grade 1-5 vs. 0). RNA-specific gene targets implicated in PC were obtained via miRTarBase, tRFtarget, and KEGG. Among 34 patients receiving an MRI-guided biopsy (26% AA, 74% White; median age 64), 53% had cancer at MRI-indicated target sites (44% in AA, 56% in White), with 27% grade 1 (33% in AA, 24% in White), 20% grade 2-3 (11% in AA, 24% in White), and 6% grade 4-5 cancer (0% in AA, 8% in White). Multivariate analysis across all samples revealed 20 miRs with significantly increased expression in grade 4-5 vs. grade 1 cancer (p=0.001-0.042), two of which also had increased expression in grades 2-3 (p=0.026; miR-3529-3p, miR-7-5p). miR gene targets (n= 320) included PTEN (miR-130b-3p, miR-182-5p, miR-200a-3p, miR-20b-5p), CDKN1B (miR-200b-3p), and KLF6 (miR-20b-5p) associated with grade 4-5; and AR (miR-7-5p) associated with grade 2-3 cancer. Seven tRFs had increased expression in grade 2-3 vs. 1 cancer (p=0.0003-0.047). Of note, Mi-tRNA-Asp-GTC-2-1_L22_pos29 targeted KLF6, MAD1L1, and ZFHX3 genes. Nine tRFs had decreased expression in grade 2-3 vs. 1 cancer (p=0.003-0.33), of which 5-tRNA-Lys-CTT-2-1_L30, S3-mito-tRNA-Val-TAC_L22, and S5-tRNA-Gly-TCC-1-1_L30 targeted MAD1L1, RNASEL, ZFHX3, AR, EHBP1, KLF6, and PTEN. Two tRFs decreased in expression with lower age at biopsy (p=0.036-0.046). When stratified by race, two miR (miR-941, p=0.025; miR-140-5p, p=0.009) and five tRF (p=0.0003-0.037) had increased expression with positive cancer status uniquely among AA men. Of these, M5-tRNA-Glu-CTC-1-1_L23 and Mi-tRNA-Val-CAC-5-1_L20_pos56 had increased and Mi-tRNA-Val-CAC-5-1_L21_pos55 and Mi-tRNA-Asp-GTC-2-1_L23_pos51 had decreased expression in grade 2-3 vs. 1 cancer (p<0.001; gene targets AR, PTEN). The small sample size precluded additional comparisons. Our pilot study revealed differential miR and tRF expression across risk and self-reported race with known PC gene targets, highlighting their potential as biomarkers for future investigation in the context of persistent racial disparities in PC outcomes. Citation Format: Yeonju Kim, Gregory Dyson, Alan Dombkowski, Hunter Dlugas, Steven Lucas, Jason Domina, Cathryn Bock. MicroRNA and tRNA fragments as biomarkers for prostate cancer aggressiveness in self-reported black and white men: Results of a pilot study [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C018.

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction.

Supplementary Table 1 from Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Background and Objectives: Bronchopulmonary dysplasia (BPD) is a serious complication of preterm birth, resulting in significant morbidity and mortality. Recent studies have suggested that microRNA (miRNA) dysregulation is involved in the pathogenesis of BPD and may serve as biomarkers for early detection. We conducted a directed search for dysregulated miRNAs in lung and heart autopsy samples of infants with histologic BPD. Methods: We used archived lung and heart samples from BPD (13 lung, 6 heart) and control (24 lung, 5 heart) subjects. To measure miRNA expression, RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue specimens then reverse-transcribed, labeled, and hybridized to miRNA microarrays. The microarrays were scanned, and data were quantile normalized. Statistical analysis with a moderated t-test and control of the false discovery rate (5%) was used to compare normalized miRNA expression values between clinical categories. Results: With our set of 48 samples, 43 miRNAs had a significant difference in expression comparing BPD to non-BPD controls. Among the most statistically significant miRNAs, miR-378b, miRNA-184, miRNA-3667-5p, miRNA-3976, miRNA-4646-5p, and miRNA-7846-3p were all consistently upregulated in both the heart and lung tissues of BPD subjects. The cellular pathway predicted to be most affected by these miRNAs is the Hippo signaling pathway. Conclusions: This study identifies miRNAs that are similarly dysregulated in postmortem lung and heart samples in subjects with histologic BPD. These miRNAs may contribute to the pathogenesis of BPD, have potential as biomarkers, and may provide insight to novel approaches for diagnosis and treatment.

Abstract Background: The majority of lung cancer patients are diagnosed at an advanced or metastatic stage and their 5-year survival rate is < 5%. However, when patients are diagnosed at an early stage, Stage I, they have ~70% 5-year survival rate. The aim of the study was to identify Stage I lung cancer miRNA biomarkers for early lung cancer detection. Methods: RNA was isolated from serum of 11 healthy subjects and 45 non-small cell lung cancer (NSCLC) patients comprised of 21 Stage I, 8 Stage II and 11 Stage III adenocarcinoma (AC) and 5 squamous cell carcinoma (SCC) patients, and from NSCLC (A549, H23, H1437, H460 and H1299) and kidney (ACHN) cell lines. Eight miRNA lung cancer biomarker candidates were selected based on previously published studies, were analyzed by qRT-PCR/Taqman® analyses. Results: MiR486 levels were up-regulated in Stages I, II and III AC. Whereas MiR-122 and miR-29c were not detected in serum samples in most healthy subjects, these respective miRNAs were expressed in 62% and 71% of Stage I and 88% and 100% of Stage II and 91 and 100% of Stage III lung AC patients. Ratios of miR-486/miR-16 and miR-29c/miR-16 were ~10-fold higher in A549 lung AC cell lysates and media compared with renal ACHN cell lysates and media and both miRNAs were higher in A549 cells when compared to other lung cancer cell lines. Conclusions: Upregulation of miR-486 and miR-29c in blood can be used as a biomarker for early AC lung cancer diagnosis.

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and sup-pression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the "vicious cycle" between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy.

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥ 2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment.

Asthma affects nearly 6 million children in the United States. Approximately 50% of children with asthma experience an exacerbation annually, 16.7% require an emergency department (ED) visit, and nearly 4.7% are hospitalized yearly, making asthma the third leading cause of pediatric inpatient admissions.1Zahran H.S. Bailey C.M. Damon S.A. Garbe P.L. Breysse P.N. Vital Signs: asthma in Children-United States, 2001-2016.MMWR Morb Mortal Wkly Rep. 2018; 67: 149-155Crossref PubMed Scopus (275) Google Scholar Asthma is a multifactorial disease that is often triggered by environmental factors in genetically predisposed individuals. Because the ED is frequently the first, if not the only, point of access to health care for many children with acute asthma, it provides a unique, yet largely unexplored, clinical setting to investigate novel molecular biomarkers for asthma severity and treatment response. MicroRNAs are a class of short, highly stable endogenous noncoding RNAs involved in regulating multiple cellular processes, either by preventing translation and/or inducing transcript degradation.2Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29561) Google Scholar Past research has primarily focused on identifying changes in microRNA expression associated with airway inflammation in acute asthma3Lu T.X. Munitz A. Rothenberg M.E. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression.J Immunol. 2009; 182: 4994-5002Crossref PubMed Scopus (492) Google Scholar; however, there is a paucity of pediatric data, as these studies were conducted primarily in adults, were set in subacute care settings, and utilized invasive methods of sample collection either via bronchoscopy or blood samples. We sought to evaluate microRNA expression patterns in a pediatric ED patient population with varying asthma severity by using tissue obtained via nasal swabs. Nasal and bronchial cells have identical morphology and similar responses to cytokines in vivo and in vitro4McDougall C.M. Blaylock M.G. Douglas J.G. Brooker R.J. Helms P.J. Walsh G.M. Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.Am J Respir Cell Mol Biol. 2008; 39: 560-568Crossref PubMed Scopus (128) Google Scholar; therefore, nasal epithelia could potentially constitute an accessible surrogate for studying lower airway inflammation. The main objectives of our study were to demonstrate the feasibility of obtaining adequate amounts of high-quality microRNAs via nasal swabs and to compare microRNA expression patterns in pediatric patients with varying severity of asthma. We conducted a prospective, observational study on children 18 years of age or younger and presenting to an inner city, freestanding pediatric ED. This study was approved by the institutional review board, and patients were enrolled after informed consent had been obtained. We enrolled patients in 1 of the following 4 cohorts: patients with severe asthma exacerbation; patients with moderate asthma exacerbation; patients with a history of asthma with no exacerbation, and patients with no history of asthma or atopy (Table I). We defined acute asthma as wheezing or respiratory distress in a patient with a prior diagnosis of asthma or reactive airway disease. Asthma severity was determined by using the Pediatric Asthma Severity Score, a standardized asthma assessment tool. We excluded patients with a history of heart disease, those with lung disease other than asthma, and those who refused informed consent.Table IPatient demographicsCharacteristicTotal no. (N = 39)No history of asthma (n = 10)History of asthma with no exacerbation (n = 10)Moderate asthma exacerbation (n = 10)Severe asthma exacerbation (n = 9)Sex Male237583 Female163526Age <1 y11000 1-10 y224558 >10 y165551Ethnicity White43010 African American3571099 Hispanic00000Family history Asthma293899 Eczema233677Patient history of eczema190667 Open table in a new tab We enrolled a total of 50 participants and collected nasal swabs from each patient. RNA was successfully extracted and tested for quality from 48 specimens (see Methods in this article’s Online Repository at www.jacionline.org). MicroRNA expression microarrays were performed for approximately 10 patient samples having the best RNA quality in each group (N = 39 in total; see the Methods section and see also Table E1 in this article’s Online Repository at www.jacionline.org). Initial statistical analysis revealed that the expression values of some microRNAs were dependent on RNA quality. Therefore, for subsequent statistical analysis we excluded 11 samples with an RNA quality score less than 7 (see Methods). The remaining 28 microarrays included 8 samples each from the controls and the cohort with severe asthma exacerbation and 6 samples from both the cohort with moderate asthma exacerbation and the no-exacerbation cohort. With use of stringent statistical analysis in which the probability of a false positive was restricted to 1%, comparison of the group with severe asthma exacerbation and the control group revealed that microRNA miR-582-5p is significantly induced (∼24-fold) in the cohort of those with severe asthma exacerbation (Fig 1). The expression level of miR- 582-5p in the group with moderate exacerbation and the group with no exacerbation is intermediate compared with that in the control group and the group with severe asthma exacerbation (see Fig E1 in in this article’s Online Repository at www.jacionline.org). Interestingly, miR-582-5p has been found to be upregulated in sputum of subjects exposed to ozone, altering their respiratory immune response.5Fry R.C. Rager J.E. Bauer R. Sebastian E. Peden D.B. Jaspers I et al.Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects.Am J Physiol Lung Cell Mol Physiol. 2014; 306: L1129-L1137Crossref PubMed Scopus (61) Google Scholar We investigated whether the increase in miR-582-5p expression might be due to alterations in cell populations. Using a set of microRNAs characteristic for lymphocytes, red blood cells, immune cells, and epithelial cells, we compared expression between the group with severe asthma exacerbation and the control group. No significant differences were observed (see Fig E2 and the Methods section in this article’s Online Repository at www.jacionline.org). Our study demonstrated that it is feasible to obtain adequate quantities of high-quality microRNAs by using a noninvasive approach and conduct complex genomic studies of pediatric asthma in the ED. Although the use of blood and/or bronchial epithelial cells has been a preferred method of obtaining the “ideal” tissue sample for biomarker studies in the past, this approach is not always practical, especially in the pediatric population. Here, we show that nasal epithelium can be used as a bronchial epithelium surrogate obtained via a substantially less invasive method compared with endobronchial lavage. There are several limitations to our study. The first is the small cohort size. Although prior research revealed that significant differences in gene expression are detectable in small sample sizes (N ≤ 30),6McGeachie M.J. Davis J.S. Kho A.T. Dahlin A. Sordillo J.E. Sun M. et al.Asthma remission: predicting future airways responsiveness using an miRNA network.J Allergy Clinical Immunol. 2017; 140: 598-600.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar our findings warrant further validation. However, our statistical analysis showed that significant changes in microRNA expression could be detected in nasal epithelia with a low false-positive rate, even in a limited cohort. We found that high-quality RNAs are important to obtain robust results. Further improvements in the collection protocol may provide improved consistency in RNA quality. Second, the majority of enrolled patients were African American, reflecting the demographics of our ED population. A multicenter study with a diverse population would be the optimal setting to validate our results. In conclusion, it is feasible to obtain high-quality microRNAs from nasal epithelium in pediatric patients presenting to the ED. In this proof-of-principle study, we demonstrated that miR-582-5p is robustly overexpressed in nasal epithelia of children with severe, acute asthma. Subsequent studies and validation are warranted to further investigate the relationship between miR-582-5p and asthma severity to explore the potential of this microRNA as a clinical biomarker and to help guide targeted therapies. We thank the patients and their families for their kind cooperation. We also thank Elizabeth Duffy for her research and editorial support. Patients were enrolled and samples were collected in the ED of Children’s Hospital of Michigan between June 2012 and June 2013. Nasal epithelia were collected by using a Rhino-Pro Curette (Arlington Scientific, Springville, Utah). Samples were stored at –80˚ C until they were analyzed for microRNA expression. Total RNA from nasal epithelia was isolated by using the All-in-One Purification Kit per the vendor’s protocol (Norgen Biotek Corp, Thorold, Ontario, Canada). The quantity, purity, and quality of the RNA were verified, and an RNA integrity number (RIN) was assigned to each specimen. RINs range from 1 to 10, with 10 representing the best quality. Samples with a RIN less than 7.0 (n = 11) were excluded from subsequent statistical analysis, as this reflected RNA degradation (Table E1).E1Schroeder A. Mueller O. Stocker S. Salowsky R. Leiber M. Gassmann M. et al.The RIN: an RNA integrity number for assigning integrity values to RNA measurements.BMC Molecular Biol. 2006; 7: 3Crossref PubMed Scopus (1817) Google Scholar Expression of microRNAs was measured in each sample by using microRNA expression arrays (G4872A-031181, Agilent, Santa Clara, Calif). For each sample, 100 ng of total RNA and Agilent microRNA spike-in controls were processed and fluorescently labeled per the vendor’s protocol. Hybridization was performed per the vendor protocol, and the slides were scanned with an Agilent dual-laser scanner. Tiff images were analyzed by using Agilent’s feature extraction software (version 11.0.1.1) to obtain fluorescent intensities for each spot on the arrays. Numerous quality control parameters were inspected. The data were imported into GeneSpring (Agilent) for analysis, and probe intensity values were quantile-normalized within each microarray. Low-intensity expression values are the least reliable; therefore, we first filtered out microRNA probes in the lower 20 percentile of expression of the overall microarray. This provided 1078 microRNAs and controls (of 1368) for subsequent analysis. A moderated t test (GeneSpring) was applied to the normalized expression data for the 1368 microRNAs and used to test for statistical significance between groups. The type I (false-positive) error rate was controlled by using the Westfall-Young family-wise multiple test correction. A corrected P value of .01 was considered significant. With this stringency, there is only a 1% chance of having 1 or more false positives among the microRNAs identified as significant. Fold change (FC) is often used in analysis of gene expression data to provide a measure of the change in the expression level of a gene when comparing 2 conditions. The FC for a given gene is derived from the ratio (R) of the expression level for the gene in the 2 conditions (A and B), where R equals A divided by B. Then, FC equals R when R is greater than or equal to 0, and FC equals –1 divided by R when R is less than 0. A log transformation of the expression ratio (eg, log2R) is also a convenient representation because positive values represent an increase in expression and negative values reflect a decrease.E2Wang Y. Yang L. Li P. Huang H. Liu T. He H. et al.Circulating microRNA Signatures associated with childhood asthma.Clin Lab. 2015; 61: 467-474PubMed Google Scholar Potential differences in cell type populations were investigated by examining the expression values of microRNAs that are characteristic markers of specific cell types.E3Teruel-Montoya R. Kong X. Abraham S. Ma L. Kunapuli S.P. Holinstat M. et al.MicroRNA expression differences in human hematopoietic cell lineages enable regulated transgene expression.PLoS One. 2014; 9: e102259Crossref PubMed Scopus (64) Google Scholar, E4Gilicze A.B. Wiener Z. Tóth S. Buzás E. Pállinger É. Falcone F.H. et al.Myeloid-derived microRNAs, miR-223, miR27a, and miR-652, are dominant players in myeloid regulation.Biomed Res Int. 2014; 2014: 870267Crossref PubMed Scopus (27) Google Scholar, E5Tserel L. Runnel T. Kisand K. Pihlap M. Bakhoff L. Kolde R. et al.MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of Toll-like receptor.J Biol Chem. 2011; 286: 26487-26495Crossref PubMed Scopus (110) Google Scholar, E6Martinez-Anton A. Sokolowska M. Kern S. Davis A.S. Alsaaty S. Taubenberger J.K. et al.Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2013 Sep; 49 (PMID: 23590309): 384-395Crossref PubMed Scopus (43) Google Scholar, E7McCall M.N. Kim M.S. Adil M. Patil A.H. Lu Y. Mitchell C.J. et al.Toward the human cellular microRNAome.Genome Res. 2017; 27: 1769-1781Crossref PubMed Scopus (95) Google Scholar Expression in the group with severe asthma exacerbation and in the control group were compared (Table E2).Fig E2Cell types in collected epithelia are consistent between the control group and the group with severe asthma. Expression of microRNAs characteristic for specific cells types was compared between the controls with no history (Hx) of asthma and the subjects with severe asthma. Significant differences were not observed, suggesting consistency in cell type populations in the patient specimens. RBC, Red blood cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1RNA quality and groupSample IDGroupRIN1011Severe9.71020No Hx9.11042Severe9.11007No Hx8.91016Moderate8.71021Moderate8.71030No Hx8.71010Severe8.71001Hx8.61015Hx8.51036Moderate8.51031Severe8.51002Severe81027Hx7.91034Moderate7.91005No Hx7.91013No Hx7.91008Severe7.91014Moderate7.81040No Hx7.81023Severe7.81004Hx7.71037Hx7.71041Hx7.71006No Hx7.71022No Hx7.71048Severe7.71017Moderate71018Severe6.91026Hx6.81003No Hx6.81044Hx6.71043Hx6.51033Moderate6.31012Moderate6.21029Moderate6.21039No Hx6.21038Hx5.91045Moderate5.9ID, Identifier; Hx, history; RIN, RNA integrity number. Open table in a new tab Table E2MicroRNA expression by cell typemicroRNACell typemiR-200b-3pEpitheliallet-7f-5pPlateletsmiR-451aRed blood cellsmiR-148a-3pGranulocytesmiR-150-5pLymphocytesmiR-212-3pDendritic and macrophagesmiR-99b-5pDendritic and macrophagesmiR-125a-3pMucosal mast cells Open table in a new tab ID, Identifier; Hx, history; RIN, RNA integrity number.

Fetoplacental neuroblastoma metastasis has been postulated as a mechanism accounting for concordant cases where one twin develops a primary tumour and the second twin manifests the disease without an identifiable primary site. These tumours may originate and spread concomitantly due to the same genetic background shared by monozygotic twins. This study investigated the molecular profile of stage MS neuroblastoma presenting concomitantly in monozygotic twins. Comparative genomic hybridisation (aCGH) was done for each of the twin liver tumour and peripheral blood samples at diagnosis. Comparison of copy-number variation (CNV) regions revealed a set of CNVs that were common to both tumour specimens and not apparent in the blood. The CNV signature in both twins' tumours was highly similar, suggesting a common clonal origin. Additional findings included large deletion of chromosome 10 and amplification of chromosome 17. Notably, both liver samples had amplification of a short region involving DEIN (chromosome 4q34.1). Similar CNVs strongly support a common clonal origin and metastatic spread from one twin to the other. DEIN is a long-coding RNA (IncRNA) that has been found highly expressed in stage MS neuroblastoma and is likely involved in biological processes such as cell migration and metastasis.

BACKGROUND:Neuroinflammation and toll-like receptors (TLR) of the innate immune system have been implicated in epilepsy. We previously reported high levels of microRNAs miR-142-3p and miR-223-3p in epileptogenic brain tissue resected for the treatment of intractable epilepsy in children with tuberous sclerosis complex (TSC). As miR-142-3p has recently been reported to be a ligand and activator of TLR7, a detector of exogenous and endogenous single-stranded RNA, we evaluated TLR7 expression and downstream IL23A activation in surgically resected TSC brain tissue.METHODS:Gene expression analysis was performed on cortical tissue obtained from surgery of TSC children with pharmacoresistent epilepsy. Expression of TLRs 2, 4 and 7 was measured using NanoString nCounter assays. Real-time quantitative PCR was used to confirm TLR7 expression and compare TLR7 activation, indicated by IL-23A levels, to levels of miR-142-3p. Protein markers characteristic for TLR7 activation were assessed using data from our existing quantitative proteomics dataset of TSC tissue. Capillary electrophoresis Western blots were used to confirm TLR7 protein expression in a subset of samples.RESULTS:TLR7 transcript expression was present in all TSC specimens. The signaling competent form of TLR7 protein was detected in the membrane fraction of each sample tested. Downstream activation of TLR7 was found in epileptogenic lesions having elevated neuroinflammation indicated by clinical neuroimaging. TLR7 activity was significantly associated with tissue levels of miR-142-3p.CONCLUSION:TLR7 activation by microRNAs may contribute to the neuroinflammatory cascade in epilepsy in TSC. Further characterization of this mechanism may enable the combined of use of neuroimaging and TLR7 inhibitors in a personalized approach towards the treatment of intractable epilepsy.

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

Prostate cancer is one of the most common cancers in men worldwide. Currently available diagnostic and prognostic tools for this disease, such as prostate specific antigen, suffer from lack of specificity and sensitivity, resulting in over- and misdiagnosis. Hence, there is an urgent need for clinically relevant biomarkers capable of distinguishing between aggressive and nonaggressive forms of prostate cancer to aid in stratification, management and therapeutic decisions. To address this unmet need, we investigated the patterns of expression of a panel of 68 plasma-derived microRNAs (miRNAs) in a cohort of African American (AA) and European American (EA) prostate cancer patients (n = 114). miRNA qPCR results were analyzed using in-depth statistical methods, and a bioinformatics analysis was conducted to identify potential targets of the differentially expressed miRNAs. Our data demonstrate that a new previously unreported circulating miRNA signature consisting of a combination of interacting miRNAs (miR-17/miR-192) and an independent miRNA (miR-181a) are capable of segregating aggressive and nonaggressive prostate cancer in both AA and EA patients. The interacting miRNAs outperformed independent miRNAs in identifying aggressiveness. Our results suggest that these circulating miRNAs may constitute novel biomarkers of prostate cancer aggressiveness in both races and warrant further investigation.

A rapidly growing body of evidence supports the premise that neuroinflammation plays an important role in initiating and sustaining seizures in a range of pediatric epilepsies. Clinical and experimental evidence indicate that neuroinflammation is both an outcome and a contributor to seizures. In this manner, seizures that arise from an initial insult (e.g. infection, trauma, genetic mutation) contribute to an inflammatory response that subsequently promotes recurrent seizures. This cyclical relationship between seizures and neuroinflammation has been described as a 'vicious cycle.' Studies of human tissue resected for surgical treatment of refractory epilepsy have reported activated inflammatory and immune signaling pathways, while animal models have been used to demonstrate that key inflammatory mediators lead to increased seizure susceptibility. Further characterization of the molecular mechanisms involved in this cycle may ultimately enable the development of new therapeutic approaches for the treatment of epilepsy. In this brief review we focus on key inflammatory mediators that have become prominent in recent literature of epilepsy, including newly characterized microRNAs and their potential role in neuroinflammatory signaling.

BACKGROUND: Large epidemiological studies have shown that diets high in fruits reduce the risk of esophageal squamous cell carcinoma (ESCC). OBJECTIVE: The current study investigated the effects of black raspberries (BRBs) on gene expression during the development of preneoplastic esophagi in rats. METHODS: Using a post-initiation protocol, F344 rats were injected with N-nitrosomethylbenzylamine (NMBA) and then fed either a control diet or 5% BRBs. At weeks 9, 15, and 35, we euthanized subgroups of the rats and collected preneoplastic esophagi to isolate RNA samples for DNA microarray. RESULTS: Along the development of NMBA-induced preneoplastic esophagi, NMBA injections led to differential expression of 1181 genes comparing to control rats, and dietary BRBs modulated 428 genes in esophagi from NMBA-treated rats. There are 137 common genes between 1181 and 428 gene sets, and BRBs significantly reversed the expression of 133 genes. These genes are associated with multiple gene oncology functions. BRBs induced an 8.8-fold gene enrichment on the pathway of inflammatory response and regulated 10 genes involved in this pathway. Among them, BRBs significantly reversed the expression of pro-inflammatory cytokines, such as CCL2, S100A8, and IL19. CONCLUSIONS: BRBs exhibit strong anti-inflammatory effects against NMBA-induced rat esophageal tumorigenesis.

It is often desirable to increase the stability of proteins. A method for protein stabilization that has been successful in a wide range of applications is disulfide engineering. This method entails the creation of novel disulfide bonds in proteins of interest. An effective approach towards accomplishing this goal is to analyze a structural model of the target protein to identify a residue pair that is oriented in a manner consistent with disulfide formation, then mutate the residues to cysteines. This chapter reviews computational methods that have been developed to assist in disulfide engineering. In addition, a range of successful disulfide engineering applications, from stabilization of industrial enzymes to improvements in vaccine stability and efficacy, are discussed.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in various organs and arises due to mutations in the TSC1 or TSC2 genes. TSC mutations lead to a range of neurological manifestations including epilepsy, cognitive impairment, autism spectrum disorders (ASD), and brain lesions that include cortical tubers. There is evidence that seizures arise at or near cortical tubers, but it is unknown why some tubers are epileptogenic while others are not. We have previously reported increased tryptophan metabolism measured with α[11C]-methyl-l-tryptophan (AMT) positron emission tomography (PET) in epileptogenic tubers in approximately two-thirds of patients with tuberous sclerosis and intractable epilepsy. However, the underlying mechanisms leading to seizure onset in TSC remain poorly characterized. MicroRNAs are enriched in the brain and play important roles in neurodevelopment and brain function. Recent reports have shown aberrant microRNA expression in epilepsy and TSC. In this study, we performed microRNA expression profiling in brain specimens obtained from TSC patients undergoing epilepsy surgery for intractable epilepsy. Typically, in these resections several non-seizure onset tubers are resected together with the seizure-onset tubers because of their proximity. We directly compared seizure onset tubers, with and without increased tryptophan metabolism measured with PET, and non-onset tubers to assess the role of microRNAs in epileptogenesis associated with these lesions. Whether a particular tuber was epileptogenic or non-epileptogenic was determined with intracranial electrocorticography, and tryptophan metabolism was measured with AMT PET. We identified a set of five microRNAs (miR-142-3p, 142-5p, 223-3p, 200b-3p and 32-5p) that collectively distinguish among the three primary groups of tubers: non-onset/AMT-cold (NC), onset/AMT-cold (OC), and onset/AMT-hot (OH). These microRNAs were significantly upregulated in OH tubers compared to the other two groups, and microRNA expression was most significantly associated with AMT-PET uptake. The microRNAs target a group of genes enriched for synaptic signaling and epilepsy risk, including SLC12A5, SYT1, GRIN2A, GRIN2B, KCNB1, SCN2A, TSC1, and MEF2C. We confirmed the interaction between miR-32-5p and SLC12A5 using a luciferase reporter assay. Our findings provide a new avenue for subsequent mechanistic studies of tuber epileptogenesis in TSC.

Abstract The blood prostate-specific antigen (PSA) assay is used for prostate cancer diagnosis but specificity of the assay is not satisfactory. Previously a combined PSA and GDF-15 (NAG-1) score was shown to improve specificity of prostate cancer detection. Our hypothesis was that, in prostate cancer, glycoproteins were secreted from tumor tissues into blood and induce autoimmune immunoglobulin G (auto-IgG) production and, thus, measurement of the glycan-auto-IgG in blood would improve prostate cancer diagnosis. The 24 glycan-containing microarray analyses have been carried out with plasma samples obtained from 35 prostate cancer patients and 46 healthy subjects to identify glycan-binding auto-IgG biomarker candidates by incubating the auto-IgG captured by glycans spotted on the slide with biotinylated anti-human secondary IgG/streptavidin-Alexa647. Among the 24 glycans, GlcNAc-polyacrylamide (PAA) (G09) and Fucα1-3GlcNAcβ-PAA (G24) glycans showed lower signals of the auto-IgG in the prostate cancer after quantile normalization. β-D-Galactose-PAA (G04) and L-rhamnose-PAA (G08) glycans showed higher signals of the auto-IgG in prostate cancer. No auto-IgM signal was detected. Subsequently, a 5-glycan subarray analysis was developed and lower signals of G09 and G24 glycan-binding auto-IgG were verified by the subarray analysis using 35 prostate cancer plasma samples compared with 54 controls. A higher signal of Neu5Acα2-8Neu5Acα2-8Neu5Acα-sp-PAA (G81)-binding auto-IgG in prostate cancer was detected by the subarray analysis. When the result obtained with the G81-binding auto-IgG was combined with levels of PSA and NAG-1, the prediction rate of prostate cancer increased to 86.2% from 78.2% with PSA levels alone, improving diagnostic accuracy of prostate cancer. The G81 glycan-binding auto-IgG was isolated from prostate cancer and control plasma samples using G81 glycan-affinity chromatography. Western blot analysis of the auto-IgG eluate with a secondary IgG antibody revealed that the level of the 50 kDa heavy chain of the auto-IgG obtained from the prostate cancer patient was ~3-fold higher than the control sample. The 50 kDa fragment was identified as an IgG heavy chain variable region by N-terminal sequencing (Edman's degradation). Our result demonstrated that a multiplex biomarker diagnostic consisting of glycan-binding auto-IgG, PSA and NAG-1 increased specificity and sensitivity of prostate cancer diagnosis. Supported by NCI SBIR Phase I, CA159721. Citation Format: Hyesook Kim, Julia Matzenbacher Santos, Aby Joiakim, David Kaplan, Ben Rybicki, Alan Dombkowski, David A. Putt. Improvement of prostate cancer diagnosis using a multiplex test of PSA, GDF-15 (NAG-1) and glycan-binding auto-IgG in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 690.

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.