Low Density Array Research Articles

MicroRNA Expression Profile of Purified Alveolar Epithelial Type II Cells.

Alveolar type II (ATII) cells are essential for the maintenance of the alveolar homeostasis. However, knowledge of the expression of the miRNAs and miRNA-regulated networks which control homeostasis and coordinate diverse functions of murine ATII cells is limited. Therefore, we asked how miRNAs expressed in ATII cells might contribute to the regulation of signaling pathways. We purified “untouched by antibodies” ATII cells using a flow cytometric sorting method with a highly autofluorescent population of lung cells. TaqMan® miRNA low-density arrays were performed on sorted cells and intersected with miRNA profiles of ATII cells isolated according to a previously published protocol. Of 293 miRNAs expressed in both ATII preparations, 111 showed equal abundances. The target mRNAs of bona fide ATII miRNAs were used for pathway enrichment analysis. This analysis identified nine signaling pathways with known functions in fibrosis and/or epithelial-to-mesenchymal transition (EMT). In particular, a subset of 19 miRNAs was found to target 21 components of the TGF-β signaling pathway. Three of these miRNAs (miR-16-5p, -17-5p and -30c-5p) were down-modulated by TGF-β1 stimulation in human A549 cells, and concomitant up-regulation of associated mRNA targets (BMPR2, JUN, RUNX2) was observed. These results suggest an important role for miRNAs in maintaining the homeostasis of the TGF-β signaling pathway in ATII cells under physiological conditions.

299 Oral difelikefalin improves itch and inflammatory biomarkers in atopic dermatitis subjects with moderate-to-severe pruritus

Pruritus is the most frequent and burdensome atopic dermatitis (AD) symptom and can exacerbate disease via the itch-scratch cycle. Difelikefalin (DFK), a kappa-opioid receptor (KOR) agonist is approved for moderate-to-severe pruritus in adults with CKD undergoing hemodialysis. DFK blocks pruritic signaling through peripheral sensory neurons and may have anti-inflammatory activity. We tested the impact of DFK on pruritus-related and inflammatory transcriptomes of AD patients. In a phase 2 clinical study, subjects with AD and moderate-to-severe pruritus were randomized (1:1:1:1) to receive oral twice-daily DFK (3 doses) or placebo for 12 wk. A substudy (n=40) characterized effects of DFK on pruritus- and AD-related gene expression using baseline and wk 12 skin biopsies. Gene expression was measured using RNA-sequencing and TaqMan Low-Density Array qualitative polymerase chain reaction. Data from DFK groups were pooled. DFK altered expression of multiple individual pruritus- and AD-related genes. Gene set variation analysis confirmed downregulation of pruritus-related genes (eg, IL-31, TRPV2) and the Th2 pathway following 12 wk treatment with DFK, but not placebo. Changes in gene expression of pruritus- and immune-related genes significantly correlated with skin improvement for DFK-treated subjects. DFK downregulated expression of key genes implicated in pruritus and AD inflammation. Reports demonstrate an anti-pruritic effect of DFK in AD and other chronic itch conditions. DFK is a promising therapy for AD-related pruritus and may provide anti-inflammatory benefit by impacting the itch-scratch cycle.

Comparison of multiple gene expression platforms for measuring a bladder cancer hypoxia signature.

Tumour hypoxia status provides prognostic information and predicts response to hypoxia‑modifying treatments. A previous study by our group derived a 24‑gene signature to assess hypoxia in bladder cancer. The objectives of the present study were to compare platforms for generating signature scores, identify cut‑off values for prospective studies, assess intra‑tumour heterogeneity and confirm hypoxia relevance. Briefly, RNA was extracted from prospectively collected diagnostic biopsies of muscle invasive bladder cancer (51patients), and gene expression was measured using customised Taqman Low Density Array (TLDA) cards, NanoString and Clariom S arrays. Cross‑platform transferability of the gene signature was assessed using regression and concordance analysis. The cut‑off values were the cohort median expression values. Intra‑ and inter‑tumour variability were determined in a retrospective patient cohort (n=51) with multiple blocks(2‑18) from the same tumour. To demonstrate relevance, bladder cancer cell lines were exposed to hypoxia (0.1% oxygen, 24h), and extracted RNA was run on custom TLDA cards. Hypoxia scores(HS) values showed good agreement between platforms: ClariomS vs. TLDA (r=0.72, P<0.0001; concordance73%); ClariomS vs. NanoString (r=0.84, P<0.0001; 78%); TLDA vs. NanoString (r=0.80, P<0.0001;78%). Cut‑off values were 0.047 (TLDA), 7.328 (NanoString) and 6.667 (ClariomS). Intra‑tumour heterogeneity in gene expression and HS (coefficient of variation3.9%) was less than inter‑tumour (7.9%) variability. HS values were higher in bladder cancer cells exposed to hypoxia compared with normoxia (P<0.02). In conclusion, the present study revealed that application of the 24‑gene bladder cancer hypoxia signature was platform agnostic, cut‑off values determined prospectively can be used in a clinical trial, intra‑tumour heterogeneity was low and the signature was sensitive to changes in oxygen levels invitro.

MIR146A and ADIPOQ genetic variants are associated with birth weight in relation to gestational age: a cohort study.

To evaluate the genetic variants related to polycystic ovary syndrome (PCOS) and its metabolic complications in girls born small for gestational age (SGA). Retrospective birth cohort study. We evaluated 66 women of reproductive age born at term (37-42weeks of gestational age) according to the birth weight in relation to gestational age: 26 SGA and 40 AGA (Adequate for gestational age). Anthropometric and biochemical characteristics were measured, as well as the PCOS prevalence. We analyzed 48 single nucleotide polymorphisms (SNPs) previously associated with PCOS and its comorbidities using TaqMan Low-Density Array (TLDA). miRNet and STRING databases were used to predict target and disease networks. Anthropometric and biochemical characteristics did not differ between the SGA and AGA groups, as well as insulin resistance and PCOS prevalence. Two SNPs were not in Hardy-Weinberg equilibrium, the rs2910164 (MIR146A C > G) and rs182052 (ADIPOQ G > A). The rs2910164 minor allele frequency (MAF) was increased in SGA (OR, 2.77; 95%; CI, 1.22-6.29), while the rs182052 was increased AGA (OR, 0.34; 95%; CI, 0.13 - 0.88). The alleles related to reduced miRNA-146a (C) and ADIPOQ (A) activity showed increased frequency in SGA. The mature miR-146a targets 319 genes, been the CXCR4, TMEM167A and IF144L common targets and contributes to PCOS. The ADIPOQ main protein interactions were ERP44, PPARGCIA and CDH13. The miR-146a (rs2910164) and ADIPOQ (rs182052) allelic variants are related to birth weight in SGA and may predict health-related outcomes, such as PCOS and obesity risk.

Expression of neuroblastoma-related genes in bone marrow at end of high-risk neuroblastoma therapy.

Minimal disease quantification may predict event-free survival (EFS) and overall survival (OS). We evaluated mRNA expression of five neuroblastoma-associated genes (NB5 assay) in bone marrows (BM) of patients with newly diagnosed high-risk neuroblastoma who received consistent immunotherapy. mRNA expression of CHGA, DCX, DDC, PHOX2B, and TH genes in BM of 479 patients enrolled on the immunotherapy arm of Children's Oncology Group trials ANBL0032 and ANBL0931 was evaluated using real-time polymerase chain reaction (PCR)-based TaqMan low-density array. Results from end-consolidation and end-therapy were analyzed for association with five-year EFS/OS and patient and tumor characteristics. Tests of statistical significance were two-sided. NB5 assay detected neuroblastoma-related mRNA in 222 of 286 (77.6%) of BMs obtained at end-consolidation and 188 of 304 (61.8%) at end-therapy. Any mRNA level detected in end-therapy BM correlated with significantly worse EFS (57% [49.6%-63.7%] vs 73.0% [63.5%-80.4%]; P=0.005), but not OS. Analysis limited to patients in complete response at end-therapy still found a significant difference in EFS with detectable versus not detectable NB5 assay results (58.9% [49.5%-67.1%] vs 76.6% [66.1%-84.2%]; P=0.01). End-consolidation results did not correlate with EFS or OS. Multivariable analysis determined end-therapy NB5 assay BM results (P=0.02), age at diagnosis (P=0.002), and preconsolidation response (P=0.02) were significantly associated with EFS independent of other clinical and biological parameters evaluated, including end-therapy response. If further validated in additional patient cohorts, the NB5 assay's ability to independently predict EFS from end-therapy could improve patient stratification for novel maintenance therapy trials after current end-therapy to improve outcome.

MiR-1227-3p participates in the development of fetal growth restriction via regulating trophoblast cell proliferation and apoptosis

Fetal growth restriction (FGR) is a common obstetric disease, which is harmful to the pregnant women and fetuses. It has many influencing factors, but the specific etiology is not clear. MiRNA plays an important role in the fetal growth and development. In this article, we use TaqMan Low-Density Array to screen and analyze the differently expressed miRNAs in FGR-affected placenta (n = 40) and the normal placenta (n = 40). A total of 139 abnormally expressed miRNAs in the FGR-affected placenta were identified, and miR-1227-3p was the most highly downregulated miRNA. Importantly, miR-1227-3p may promote the proliferation in HTR-8/SVneo cells, while inhibited the apoptosis of HTR-8/SVneo cells. DAVID was used to analyze the pathway enrichment of target genes of miR-1227-3p to predict its mechanism of action. Furthermore, the putative targets of miR-1227-3p were predicted using the TargetScan, PicTar, DIANA LAB, and miRWalk database. The potential expression of target genes of miR-1227-3p, including PRKAB2, AKT1, PIK3R3, and MKNK1 were significantly increased in FGR-affected placenta. Taken together, miR-1227-3p may participate in the development of FGR via regulating trophoblast cell proliferation and apoptosis by targeting genes involved in the insulin pathway. MiR-1227-3p may have a potential clinical value in the prevention and treatment of FGR, we need to study further to prove its value in the future.

Gene expression signatures in PCB-exposed Slovak children in relation to their environmental exposures and socio-physical characteristics

Our previous gene expression studies in a PCB-exposed cohort of young children in Slovakia revealed that early-life exposures to PCBs and other organochlorine compounds were associated with significant alterations across several pathogenetic pathways. The present study was undertaken to further explore the high-throughput qRT-PCR-based gene expression effects by using TaqMan low-density array (TLDA) for selected genes in a sample of 55 children from the cohort. We analyzed the transcriptional changes of 11 genes in relation to PCB and organochlorine pesticide exposure levels (including DDT, DDE, HCH, and HCB), and to BMI and ethnicity in this cohort. The results indicated an overall downregulation of expression of these genes. Maximum downregulation (in fold change) was observed in the ENTPD3 gene, and the minimum level of downregulation was in CYP2D6. As per our multinomial regression model study, downregulation of LEPR gene was significantly directly correlated with all the exposure variables. Downregulation of APC, ARNT, CYP2D6, LEPR, LRP12, and MYC genes was directly correlated with BMI (kg/m2) of the individuals. Gender-specific differences in gene expression were observed in CYP2D6 (p-value 0.0001) and LEPR (p-value 0.028), while downregulation of CYP2D6 (p-value 0.01), LEPR (p-value 0.02), LRP12 (p-value 0.04), and MYC (p-value 0.02) genes was consistently observed in Roma children compared to Caucasians. The investigation of such health disparities must be emphasized in future research, together with interventions to reduce the health consequences of PCB exposures. In this context, we emphasize the importance of biomarker-based approaches to future research on genetic susceptibility to the effects of these compounds.

Circulating Vitreous microRNA as Possible Biomarker in High Myopic Eyes with Macular Hole.

High myopia is a major cause of irreversible visual impairment globally. In the present study, we investigated the microRNA (miRNA) profile in the vitreous of macular hole (MH) and high myopic MH. We performed miRNA analysis using TaqMan® Low Density Arrays (Thermo Fisher Scientific, Waltham, MA, USA) to investigate the circulating vitreous miRNA profile from patients with MH (axial length < 26.5 mm, n = 11) and high myopic MH (axial length ≥ 26.5 mm, n = 11) who underwent pars plana vitrectomy. The vitreous inflammatory cytokine signature was examined in high myopic MH eyes using a multiplex assay. A miRNA-Array analysis revealed that let-7c was significantly up-regulated and miR-200a was significantly down-regulated in high myopic MH eyes compared to those in MH eyes. The bioinformatics analysis for up-regulated miRNA targeted gene identified 23 pathways including mitogen-activated protein kinase (MAPK) and several inflammatory signaling pathways, whereas the bioinformatics analysis for down-regulated miRNA targeted genes showed 32 enriched pathways including phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). The levels of inflammatory cytokines including IP-10, IFN-γ, and MCP-1 were significantly higher in the vitreous of high myopic MH eyes. These results suggest that specific miRNAs expressed in the vitreous may be associated with the pathological condition of high myopic MH and the above mentioned miRNAs may contribute to the development of inflammatory status in the vitreous of high myopic eyes.

Prediction ability for growth and maternal traits using SNP arrays based on different marker densities in Nellore cattle using the ssGBLUP.

This study aimed to investigate the prediction ability for growth and maternal traits using different low-density customized SNP arrays selected by informativeness and distribution of markers across the genome employing single-step genomic BLUP (ssGBLUP). Phenotypic records for adjusted weight at 210 and 450days of age were utilized. A total of 945 animals were genotyped with high-density chip, and 267 individuals born after 2008 were selected as validation population. We evaluated 11 scenarios using five customized density arrays (40k, 20k, 10k, 5k and 2k) and the HD array was used as desirable scenario. The GEBV predictions and BIF (Beef Improvement Federation) accuracy were obtained with BLUPF90 family programs. Linear regression was used to evaluate the prediction ability, inflation, and bias of GEBV of each customized array. An overestimation of partial GEBVs in contrast with complete GEBVs and increase of BIF accuracy with the density arrays diminished were observed. For all traits, the prediction ability was higher as the array density increased and it was similar with customized arrays higher than 10k SNPs. Level of inflation was lower as the density array increased of and was higher for MW210 effect. The bias was susceptible to overestimation of GEBVs when the density customized arrays decreased. These results revealed that the BIF accuracy is sensible to overestimation using low-density customized arrays while the prediction ability with least 10,000 informative SNPs obtained from the Illumina BovineHD BeadChip shows accurate and less biased predictions. Low-density customized arrays under ssGBLUP method could be feasible and cost-effective in genomic selection.

EICOSATETRAYNOIC ACID REGULATES MITOCHONDRIAL AND EXTRA-CELLULAR MATRIX GENE EXPRESSION AND TISSUE STIFFNESS IN A HUMAN INTESTINAL ORGANOID MODEL OF ILEAL CROHN’S DISEASE

Abstract BACKGROUND Mutations in the DUOX2 intestinal epithelial cell NADPH oxidase are associated with risk for Crohn’s Disease (CD), and may influence wound healing and the development of strictures. Patients who develop strictures exhibit reduced expression of genes encoding mitochondrial sub-units, and increased expression of genes encoding extra-cellular matrix proteins, in the ileum at diagnosis. We conducted a perturbagen bioinformatics analysis which predicted that the cyclooxygenase and lipoxygenase inhibitor eicosatetraynoic-acid (ETYA) would reverse the ileal gene signature associated with stricture development. In the current study we tested effects of ETYA in a human intestinal organoid model (HIO) system. METHODS HIO were derived from wild type (WT) and DUOX2 mutant (DUOX2var) induced pluripotent stem cells (iPSCs) prepared from CD patients. WT and DUOX2var HIO were examined under basal conditions, and following exposure to ETYA for 72 hours or 12 days. Superoxide production in EPCAM+ epithelial cells and CD90+ stromal cells was measured by flow cytometry. RNA was prepared and expression of mitochondrial and extra-cellular matrix genes linked to strictures in patients was determined using RNA sequencing and a TaqMan Low Density Array (TLDA) card. HIO tissue stiffness was measured using atomic force microscopy (AFM). RESULTS ETYA reduced superoxide production by HIO EPCAM+ epithelial cells only in WT organoids; no effect was observed in DUOX2var HIO. This was specific, as no effect of ETYA upon superoxide production was detected in CD90+ stromal cells. Under basal conditions, RNA sequencing demonstrated that WT HIO expressed core mitochondrial and extra-cellular matrix genes and enriched biologic functions implicated in CD strictures. Expression of extra-cellular matrix and wound healing genes was increased in DUOX2var HIO under basal conditions. ETYA up-regulated expression of the COX5B, POLG2, and SLC25A27 mitochondrial genes associated with lower rates of strictures, while reducing expression of the ACTA2, VIM, and COL1A1 extra-cellular matrix genes associated with higher rates of strictures, independent of DUOX2 genotype. WT and DUOX2var HIO exhibited tissue stiffness comparable to normal human ileum under basal conditions; this was significantly reduced by ETYA exposure only in DUOX2var HIO. CONCLUSIONS ETYA regulates mitochondrial and extra-cellular matrix genes implicated in stricture formation in an HIO model system. DUOX2var HIO exhibit increased extra-cellular matrix gene expression under basal conditions, which is reduced by ETYA exposure in conjunction with a reduction in tissue stiffness. Collectively, these data confirm that HIO provide a relevant model system to study mechanisms regulating stricture formation in CD, including screening of small molecules prioritized by perturbagen bioinformatics analysis.

Identification and validation of plasma biomarkers for diagnosis of breast cancer in South Asian women

Breast cancer is the most common malignancy among women globally. Development of a reliable plasma biomarker panel might serve as a non-invasive and cost-effective means for population-based screening of the disease. Transcriptomic profiling of breast tumour, paired normal and apparently normal tissues, followed by validation of the shortlisted genes using TaqMan® Low density arrays and Quantitative real-time PCR was performed in South Asian women. Fifteen candidate protein markers and 3 candidate epigenetic markers were validated first in primary breast tumours and then in plasma samples of cases [N = 202 invasive, 16 DCIS] and controls [N = 203 healthy, 37 benign] using antibody array and methylation specific PCR. Diagnostic efficiency of single and combined markers was assessed. Combination of 6 protein markers (Adipsin, Leptin, Syndecan-1, Basic fibroblast growth factor, Interleukin 17B and Dickopff-3) resulted in 65% sensitivity and 80% specificity in detecting breast cancer. Multivariate diagnostic analysis of methylation status of SOSTDC1, DACT2, WIF1 showed 100% sensitivity and up to 91% specificity in discriminating BC from benign and controls. Hence, combination of SOSTDC1, DACT2 and WIF1 was effective in differentiating breast cancer [non-invasive and invasive] from benign diseases of the breast and healthy individuals and could help as a complementary diagnostic tool for breast cancer.

MicroRNAs transcriptional profiling of mammary stem cells isolated by PKH26 staining and FACS sorting.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform.

Whole-transcriptome analysis in acute lymphoblastic leukemia: a report from the DFCI ALL Consortium Protocol 16-001

AbstractThe molecular hallmark of childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent, prognostic genetic alterations, many of which are cryptic by conventional cytogenetics. RNA sequencing (RNA-seq) is a powerful next-generation sequencing technology that can simultaneously identify cryptic gene rearrangements, sequence mutations and gene expression profiles in a single assay. We examined the feasibility and utility of incorporating RNA-seq into a prospective multicenter phase 3 clinical trial for children with newly diagnosed ALL. The Dana-Farber Cancer Institute ALL Consortium Protocol 16-001 enrolled 173 patients with ALL who consented to optional studies and had samples available for RNA-seq. RNA-seq identified at least 1 alteration in 157 patients (91%). Fusion detection was 100% concordant with results obtained from conventional cytogenetic analyses. An additional 56 gene fusions were identified by RNA-seq, many of which confer prognostic or therapeutic significance. Gene expression profiling enabled further molecular classification into the following B-cell ALL (B-ALL) subgroups: high hyperdiploid (n = 36), ETV6-RUNX1/-like (n = 31), TCF3-PBX1 (n = 7), KMT2A-rearranged (KMT2A-R; n = 5), intrachromosomal amplification of chromosome 21 (iAMP21) (n = 1), hypodiploid (n = 1), Philadelphia chromosome (Ph)-positive/Ph-like (n = 16), DUX4-R (n = 11), PAX5 alterations (PAX5 alt; n = 11), PAX5 P80R (n = 1), ZNF384-R (n = 4), NUTM1-R (n = 1), MEF2D-R (n = 1), and others (n = 10). RNA-seq identified 141 nonsynonymous mutations in 93 patients (54%); the most frequent were RAS-MAPK pathway mutations. Among 79 patients with both low-density array and RNA-seq data for the Philadelphia chromosome-like gene signature prediction, results were concordant in 74 patients (94%). In conclusion, RNA-seq identified several clinically relevant genetic alterations not detected by conventional methods, which supports the integration of this technology into front-line pediatric ALL trials. This trial was registered at www.clinicaltrials.gov as #NCT03020030.

In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium – The framework for functional studies on airway differentiation in ciliopathies

Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells.Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.

The influence of sleep-wake rhythms on AD-related pathology in 3xTgAD mice.

Age-associated fragmentation of the daily sleep-wake rhythm is believed to be significant in the development of AD. While it is known that sleep deprivation can worsen AD-related pathology, such as amyloid (Aβ) deposition and neuroinflammation, the effects of fragmentation of the daily sleep-wake rhythm are unknown. It is possible that sleep fragmentation (SF), as a less stressful approach to the manipulation of the sleep-wake cycle, could better model AD-related sleep disturbances. We tested this hypothesis by subjecting female 3x-TgAD mice to a chronic SF protocol for a period of 4 weeks. SF consisted of 4 daily x 1 hour sleep disruptions, evenly interspersed throughout the light phase. During SF, mice were kept awake with novel objects and gently stimulated with a paintbrush; control mice were left undisturbed (US) during these periods. Mice were individually housed in PiezoSleep cages (Signal Solutions LLC) for sleep recordings during the first and fourth weeks of the study and group-housed in regular cages during the second and third weeks. Per 24 hours, SF mice exhibited significantly lower levels of sleep in the light phase (∼18% reduction) and higher levels of sleep in the dark phase (∼15% increase), with only a modest decrease (∼4%) in the total amount of daily sleep. After SF, Aβ40 and Aβ42 levels were significantly higher in the RIPA soluble fraction, as assayed by ELISA. An examination of mRNA using TaqMan low-density gene expression arrays indicated that markers of both microglial activation (ctsd, cst7, and clec7a) and proinflammatory cytokines (TNFα, IL6, and IL-1β) were elevated in mice subjected to SF as compared to the US group. Interestingly, the increase in Aβ and markers of neuroinflammation found in the hippocampus was not observed in the cortex, indicating at least some degree of neuroanatomical specificity in the effects of SF. These findings demonstrate that a protocol for fragmenting the daily sleep-wake rhythm in 3x-TgAD mice can impact the development of AD-related pathology. These studies also support the concept that improving sleep consolidation in individuals at risk for AD may be beneficial for slowing the onset or progression of AD.

Extracellular vesicle miRNA from cerebrospinal fluid as potential biomarkers for neurodegeneration

AbstractBackgroundExtracellular vesicles (EVs) are a diverse group of small membranous particles classified by protein and nucleic acid content, size, method of biogenesis, and cellular origin. The need for predictive biomarkers for neurodegenerative disease is critical for early detection and potential therapeutic intervention. We have previously identified multiple high ranking miRNAs in total cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD). To investigate whether these miRNAs are released from cells within EVs, we developed a pipeline to isolate and analyze EV miRNA.MethodEVs are isolated by size exclusion chromatography (SEC) from CSF collected from living donors. EV counts and sizes are determined by nanoparticle tracking analysis (NTA). Immunoblots are used to characterize EV composition and assess purity. Transmission electron microscopy (TEM) is used to assess size and EV structural characteristics. EV total RNA is isolated using commercial based methods. Quantitative PCR (qPCR) is performed using TaqMan Low Density Array (TLDA) miRNA cards.ResultThe CSF EV isolation protocol developed here is capable of isolating pure vesicles of the appropriate size and protein composition. Following miRNA qPCR, more than half of the high ranking miRNAs identified in total CSF from AD patients were observed in the isolated EV population.ConclusionThis pipeline is suitable for isolating EVs and assessing miRNA content in lumbar CSF. A large number of miRNAs isolated from EVs appear to correlate well with previously published data from total CSF, indicating a potential role for EV miRNA cargo as biomarkers for neurodegenerative diseases such as AD.

Endogenous control miRNAs for normalization in plasma samples of Alzheimer’s disease patients from a discovery and validation cohort

AbstractBackgroundSeveral studies had explored the role of miRNAs in Alzheimer’s disease (AD). The identification of endogenous controls (ECs) would contribute to the standardization of these biomarkers in AD but they have not yet been established. The objective of the study was to identify miRNAs that can be used as ECs in AD.MethodWe evaluated 145 patients divided into two different cohorts. One was a discovery cohort of 19 women diagnosed with mild to moderate AD (Mini‐Mental State Examination (MMSE) score ≥ 20) and with confirmed pathologic levels of Aβ42 in CSF. The stability assessment cohort consisted of 126 individuals: 24 control subjects, 25 subjects with MCI and negative for CSF biomarkers, 22 subjects with MCI and positive for CSF biomarkers and 55 subjects with AD and positive for CSF biomarkers.In the discovery cohort, a profile of 384 miRNAs was determined in plasma by TaqMan Low Density Array. The best EC candidates were identified by mean‐centering and concordance correlation restricted normalization methods. The stability of the EC candidates was assessed using the GeNorm, BestKeeper and NormFinder algorithms.ResultNine miRNAs (hsa‐miR‐324‐5p, hsa‐miR‐22‐5p, hsa‐miR‐103a‐2‐5p, hsa‐miR‐362‐5p, hsa‐miR‐425‐3p, hsa‐miR‐423‐5p, hsa‐let‐7i‐3p, hsa‐miR‐532‐5p, and hsa‐miR‐1301‐3p) were identified as EC candidates in the discovery cohort. The validation results indicated that hsa‐miR‐103a‐2‐5p was the best EC, followed by hsa‐miR‐22‐5p, hsa‐miR‐1301‐3p, and hsa‐miR‐425‐3p, which had similar stability values in all three algorithms.ConclusionWe identified a profile of four miRNAs as potential plasma ECs to be used for normalization of miRNA expression data in studies of subjects with cognitive impairment.

Low-density arrays of ultra-small InAs nanostructures obtained by two-stage arsenic exposure during droplet epitaxy

In order to consider quantum dots as single objects and to use them in modern electronic and photonic devices, they must be well-isolated from each other and have an appropriate size and structural quality. This is a big challenge that is difficult to achieve with traditional technological methods, such as the Stranski-Krastanov growth mechanism. In this paper, we present a novel droplet epitaxial technique for the fabrication of small-sized (∼25 nm) InAs/GaAs nanostructures with a low surface density (<1∙108cm−2). To achieve this result, we develop a growth method based on two-stage crystallization in the arsenic flux. At the first stage, the droplet size is reduced by spreading the droplet material over the surface due to the diffusion decay of droplets in an ultra-low arsenic flux. At the second stage, crystallization is carried out in a large arsenic flux while heating the substrate in order to fix the size and shape of nanodroplets and prevent them from further decaying. We demonstrate that the size dispersion of nanostructures is small and the process is well-reproducible. Thus, the presented approach makes it possible to obtain low-density quantum dots with an ultra-small size required for advanced optical applications.

Philadelphia-like acute lymphoblastic leukemia: Characterization in a pediatric cohort in a referral center in Colombia.

BackgroundPhiladelphia‐like (Ph‐like) acute lymphoblastic leukemia (ALL) is a subtype of pediatric leukemia with high risk factors and poor outcome. There are few reports of its prevalence in Latin America.AimThis study evaluated the frequency and clinical and biological characteristics of Ph‐like ALL in a pediatric cancer center in Colombia.MethodsThe Ph‐like genetic profile was analyzed by a low‐density array (LDA). Samples from patients with Ph‐like ALL were analyzed by fluorescent in situ hybridization for cytokine receptor like factor 2 (CRLF2) and ABL proto‐oncogene 1, non‐receptor tyrosine kinase (ABL1) rearrangements. Copy number variations were assessed by multiplex ligation probe amplification.ResultsData from 121 patients were analyzed. Fifteen patients (12.4%) had Ph‐like ALL, and these patients had significantly higher leukocyte counts at diagnosis and higher levels of minimal residual disease on days 15 and 33 of induction than patients without the Ph‐like subtype. There were no significant differences in sex, age, or response to prednisone at day 8 between the two groups. CRLF2 rearrangements were identified in eight patients, and ABL1 rearrangements were identified in two patients. Other genetic alterations alone or in combination were identified in 77% of patients, including deletions in cyclin dependent kinase inhibitor 2 A/B (46.2%), IKAROS family zinc finger 1 (38.3%), and paired box 5 (30.8%).ConclusionsPh‐like ALL had a 12.4% prevalence in our cohort of patients with pediatric ALL. The identification of this group of patients has importance for risk stratification and future targeted therapy.

Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance

Transforming growth factor-beta 1 (TGF-β1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-β1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-β1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-β1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-β1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.