LNCRNA expression landscape and specificity between brain regions

Long noncoding RNAs (lncRNAs) are transcribed into low potential protein coding RNA molecules, which account for over 70% of mammalian transcriptional products. The role of lncRNAs and their expression is still largely unknown, and the subject of recent investigations. Here, we used bulk RNA sequencing data from the Genotype-Tissue Expression (GTEx) project to reveal the occurrence and identify the specificity of lncRNAs in 13 brain regions (1000 samples). We observed that these highly specific lncRNA were co-expressed with previously known mRNA markers for the 13 study regions of the brain. Further investigation revealed that splicing could influence the divergent biogenesis and enrichment of specific lncRNA alleles in different brain regions. Overall, we demonstrate the use of lncRNA as an independent tool for deconvolving brain regions and further highlights its use for cell-type identification from bulk transcriptome data.

Adropin correlates with aging-related neuropathology in humans and improves cognitive function in aging mice

The neural functions of adropin, a secreted peptide highly expressed in the brain, have not been investigated. In humans, adropin is highly expressed in astrocytes and peaks during critical postnatal periods of brain development. Gene enrichment analysis of transcripts correlating with adropin expression suggests processes relevant to aging-related neurodegenerative diseases that vary with age and dementia state, possibly indicating survivor bias. In people aged <40 y and ‘old-old’ (>75 y) diagnosed with dementia, adropin correlates positively with genes involved in mitochondrial processes. In the ‘old-old’ without dementia adropin expression correlates positively with morphogenesis and synapse function. Potent neurotrophic responses in primary cultured neurons are consistent with adropin supporting the development and function of neural networks. Adropin expression in the ‘old-old’ also correlates positively with protein markers of tau-related neuropathologies and inflammation, particularly in those without dementia. How variation in brain adropin expression affects neurological aging was investigated using old (18-month) C57BL/6J mice. In mice adropin is expressed in neurons, oligodendrocyte progenitor cells, oligodendrocytes, and microglia and shows correlative relationships with groups of genes involved in neurodegeneration and cellular metabolism. Increasing adropin expression using transgenesis improved spatial learning and memory, novel object recognition, resilience to exposure to new environments, and reduced mRNA markers of inflammation in old mice. Treatment with synthetic adropin peptide also reversed age-related declines in cognitive functions and affected expression of genes involved in morphogenesis and cellular metabolism. Collectively, these results establish a link between adropin expression and neural energy metabolism and indicate a potential therapy against neurological aging.

Engineered human meniscus’ matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions

Low oxygen and mechanical loading may play roles in regulating the fibrocartilaginous phenotype of the human inner meniscus, but their combination in engineered tissues remains unstudied. Here, we investigated how continuous low oxygen (“hypoxia”) combined with dynamic compression would affect the fibrocartilaginous “inner meniscus-like” matrix-forming phenotype of human meniscus fibrochondrocytes (MFCs) in a porous type I collagen scaffold. Freshly-seeded MFC scaffolds were cultured for 4 weeks in either 3 or 20% O2 or pre-cultured for 2 weeks in 3% O2 and then dynamically compressed for 2 weeks (10% strain, 1 Hz, 1 h/day, 5 days/week), all with or without TGF-β3 supplementation. TGF-β3 supplementation was found necessary to induce matrix formation by MFCs in the collagen scaffold regardless of oxygen tension and application of the dynamic compression loading regime. Neither hypoxia under static culture nor hypoxia combined with dynamic compression had significant effects on expression of specific protein and mRNA markers for the fibrocartilaginous matrix-forming phenotype. Mechanical properties significantly increased over the two-week loading period but were not different between static and dynamic-loaded tissues after the loading period. These findings indicate that 3% O2 applied immediately after scaffold seeding and dynamic compression to 10% strain do not affect the fibrocartilaginous matrix-forming phenotype of human MFCs in this type I collagen scaffold. It is possible that a delayed hypoxia treatment and an optimized pre-culture period and loading regime combination would have led to different outcomes.

Specific and Sensitive Detection of Neuroblastoma mRNA Markers by Multiplex RT-qPCR

mRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.

Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy

Earlier detection of progression risk in diabetic nephropathy will allow earlier intervention to reduce progression. The hypothesis that urinary pellet podocyte mRNA is a more sensitive progression risk marker than microalbuminuria was tested. A cross sectional cohort of 165 type 2 diabetics and 41 age and sex-matched controls were enrolled. Podocyte stress (Urinary pellet podocin:nephrin mRNA ratio), podocyte detachment (Urinary pellet podocin mRNA:creatinine ratio: UPPod:CR) and a tubular marker (Urinary pellet aquaporin 2:creatinine ratio) were measured in macro-albuminuric, micro-albuminuric and norm-albuminuric groups. eGFR was reassessed after 4 years in 124 available diabetic subjects. Urinary pellet podocyte and tubular mRNA markers were increased in all diabetic groups in cross-sectional analysis. After 4 years of follow-up univariable and multivariate model analysis showed that the only urinary markers significantly related to eGFR slope were UPPod:CR (P < 0.01) and albuminuria (P < 0.01). AUC analysis using K-fold cross validation to predict eGFR loss of ≥ 3 ml/min/1.73m2/year showed that UPPod:CR and albuminuria each improved the AUC similarly such that combined with clinical variables they gave an AUC = 0.70. Podocyte markers and albuminuria had overlapping AUC contributions, as expected if podocyte depletion causes albuminuria. In the norm-albuminuria cohort (n = 75) baseline UPPod:CR was associated with development of albuminuria (P = 0.007) and, in the tertile with both normal kidney function (eGFR 84 ± 11.7 ml/min/1.73m2) and norm-albuminuria at baseline, UPPod:CR was associated with eGFR loss rate (P = 0.003). In type 2 diabetics with micro- or macro-albuminuria UPPod:CR and albuminuria were equally good at predicting eGFR loss. For norm-albuminuric type 2 diabetics UPPod:CR predicted both albuminuria and eGFR loss.

Signature mRNA markers in extracellular vesicles for the accurate diagnosis of colorectal cancer

Background With the increasing incidence of colorectal cancer (CRC), its accurate diagnosis is critical and in high demand. However, conventional methods are not ideal due to invasiveness and low accuracy. Herein, we aimed to identify efficient CRC mRNA markers in a non-invasive manner using CRC-derived extracellular vesicles (EVs). The expression levels of EV mRNAs from cancer cell lines were compared with those of a normal cell line using quantitative polymerase chain reaction. Eight markers were evaluated in plasma EVs from CRC patients and healthy controls. The diagnostic value of each marker, individually or in combination, was then determined using recessive operating characteristics analyses and the Mann-Whitney U test. Results Eight mRNA markers (MYC, VEGF, CDX2, CD133, CEA, CK19, EpCAM, and CD24) were found to be more abundant in EVs derived from cancer cell lines compared to control cell lines. A combination of VEGF and CD133 showed the highest sensitivity (100%), specificity (80%), and accuracy (93%) and an area under the curve of 0.96; hence, these markers were deemed to be the CRC signature. Moreover, this signature was found to be highly expressed in CRC-derived EVs compared to healthy controls. Conclusions VEGF and CD133 mRNAs comprise a unique CRC signature in EVs that has the potential to act as a novel, non-invasive, and accurate biomarker that would improve the current diagnostic platform for CRC, while also serving to strengthen the value of EV mRNA as diagnostic markers for myriad of diseases.

Markers of A1 astrocytes stratify to molecular sub-types in sporadic Creutzfeldt–Jakob disease brain

Astrocytes are glial cells of the central nervous system that become reactive under conditions of stress. The functional properties of reactive astrocytes depend on their stimulus that induces the upregulation of specific genes. Reactive astrocytes are a neuropathological feature of prion disorders; however, their role in the disease pathogenesis is not well understood. Here, we describe our studies of one polarization state of reactive astrocytes, termed A1 astrocytes, in the frontal cortex region of 35 human sporadic Creutzfeldt–Jakob disease brains encompassing a range of molecular sub-types. Examination of two mRNA markers of A1 astrocytes, C3 and GBP2, revealed a strong linear correlation between the two following their log-normalization (P = 0.0011). Both markers were found upregulated in the sporadic Creutzfeldt–Jakob disease brain compared with age-matched control tissues (P = 0.0029 and 0.0002, for C3log and GBP2log, respectively), and stratifying samples based on codon 129 genotype revealed that C3log is highest in homozygous methionine and lowest in homozygous valine patients, which followed a linear trend (P = 0.027). Upon assessing other disease parameters, a significant positive correlation was found between GBP2log and disease duration (P = 0.031). These findings provide evidence for a divergence in the astrocytic environment amongst patients with sporadic Creutzfeldt–Jakob disease based on molecular sub-type parameters of disease. While more research will be needed to determine the global changes in the genomic profiles and resulting functional properties of reactive astrocytes in disease, considering the evidence demonstrating that A1 astrocytes harbour neurotoxic properties, the changes seen in C3log and GBP2log in the current study may reflect differences in pathogenic mechanisms amongst the sporadic Creutzfeldt–Jakob disease sub-types associated with the A1 polarization state.

Mesenchymal Neuroblastoma Cells Are Undetected by Current mRNA Marker Panels: The Development of a Specific Neuroblastoma Mesenchymal Minimal Residual Disease Panel

PURPOSE Patients with neuroblastoma in molecular remission remain at considerable risk for disease recurrence. Studies have found that neuroblastoma tissue contains adrenergic (ADRN) and mesenchymal (MES) cells; the latter express low levels of commonly used markers for minimal residual disease (MRD). We identified MES-specific MRD markers and studied the dynamics of these markers during treatment. PATIENTS AND METHODS Microarray data were used to identify genes differentially expressed between ADRN and MES cell lines. Candidate genes were then studied using real-time quantitative polymerase chain reaction in cell lines and control bone marrow and peripheral blood samples. After selecting a panel of markers, serial bone marrow, peripheral blood, and peripheral blood stem cell samples were obtained from patients with high-risk neuroblastoma and tested for marker expression; survival analyses were also performed. RESULTS PRRX1, POSTN, and FMO3 mRNAs were used as a panel for specifically detecting MES mRNA in patient samples. MES mRNA was detected only rarely in peripheral blood; moreover, the presence of MES mRNA in peripheral blood stem cell samples was associated with low event-free survival and overall survival. Of note, during treatment, serial bone marrow samples obtained from 29 patients revealed a difference in dynamics between MES mRNA markers and ADRN mRNA markers. Furthermore, MES mRNA was detected in a higher percentage of patients with recurrent disease than in those who remained disease free (53% v 32%, respectively; P = .03). CONCLUSION We propose that the markers POSTN and PRRX1, in combination with FMO3, be used for real-time quantitative polymerase chain reaction–based detection of MES neuroblastoma mRNA in patient samples because these markers have a unique pattern during treatment and are more prevalent in patients with poor outcome. Together with existing markers of MRD, these new markers should be investigated further in large prospective studies.