microRNA changes in liver tissue associated with fibrosis progression in patients with hepatitis C.

BACKGROUND & AIMS: Accumulating evidence indicates that microRNAs play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis C infection. Our goal is to add to the accruing information regarding microRNA deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression. METHODS: We used next generation sequencing to profile all detectable microRNAs in liver tissue and serum from patients with hepatitis C, stages F1-F4 of fibrosis. RESULTS: We found altered expression of several microRNAs, in particular, miR-182, miR199a-5p, miR-200a-5p and miR-183 were found to be significantly upregulated in tissue from liver biopsies of hepatitis C patients with advanced fibrosis, stage F3 and F4, when compared with liver biopsies from patients with early fibrosis, stages F1 and F2. We also found miR-148-5p, miR-1260b, miR-122-3p and miR-378i among the microRNAs most significantly down-regulated from early to advanced fibrosis of the liver. We also sequenced the serum microRNAs; however, we were not able to detect significant changes in circulating microRNAs associated with fibrosis stage after adjusting for multiple tests. CONCLUSIONS: Adding measurements of tissue microRNAs acquired during routine biopsies will continue to increase our knowledge of underlying mechanisms of fibrosis. Our goal is that these data, in combination with studies from other researchers and future long-term studies, could be used to enhance the staging accuracy of liver biopsies and expand the surveillance of patients at increased risk for cancer and progression to advanced fibrosis.

Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing.

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

Label-free microscopic assessment of glioblastoma biopsy specimens prior to biobanking [corrected].

Glioblastoma is the most common primary brain tumor with a median 12- to 15-month patient survival. Improving patient survival involves better understanding the biological mechanisms of glioblastoma tumorigenesis and seeking targeted molecular therapies. Central to furthering these advances is the collection and storage of surgical biopsies (biobanking) for research. This paper addresses an imaging modality, confocal reflectance microscopy (CRM), for safely screening glioblastoma biopsy samples prior to biobanking to increase the quality of tissue provided for research and clinical trials. These data indicate that CRM can immediately identify cellularity of tissue biopsies from animal models of glioblastoma. When screening fresh human biopsy samples, CRM can differentiate a cellular glioblastoma biopsy from a necrotic biopsy without altering DNA, RNA, or protein expression of sampled tissue. These data illustrate CRM's potential for rapidly and safely screening clinical biopsy samples prior to biobanking, which demonstrates its potential as an effective screening technique that can improve the quality of tissue biobanked for patients with glioblastoma.

Differential response of C9orf72 transcripts following neuronal depolarization.

The (G4C2)n nucleotide repeat expansion (NRE) mutation in C9orf72 is the most common genetic cause of ALS and FTD. The biological functions of C9orf72 are becoming understood, but it is unclear if this gene is regulated in a neural-specific manner. Neuronal activity is a crucial modifier of biological processes in health and neurodegenerative disease contexts. Here, we show that prolonged membrane depolarization in healthy human iPSC-cortical neurons leads to a significant downregulation of a transcript variant 3 (V3) of C9orf72, with a concomitant increase in variant 2 (V2), which leads to total C9orf72 RNA transcript levels remaining unchanged. However, the same response is not observed in cortical neurons derived from patients with the C9-NRE mutation. These findings reveal the impact of depolarization on C9orf72 transcripts, and how this response diverges in C9-NRE-carriers, which may have important implications in the underlying unique clinical associations of C9-NRE transcripts and disease pathogenesis.

Breakdown of the central synapses in C9orf72-linked ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease that leads to the death of motor and cortical neurons. The clinical manifestations of ALS are heterogenous, and efficacious treatments to significantly slow the progression of the disease are lacking. Cortical hyper-excitability is observed pre-symptomatically across disease-causative genetic variants, as well as in the early stages of sporadic ALS, and typically precedes motor neuron involvement and overt neurodegeneration. The causes of cortical hyper-excitability are not yet fully understood but is mainly agreed to be an early event. The identification of the nucleotide repeat expansion (GGGGCC)n in the C9ORF72 gene has provided evidence that ALS and another neurodegenerative disease, frontotemporal dementia (FTD), are part of a disease spectrum with common genetic origins. ALS and FTD are diseases in which synaptic dysfunction is reported throughout disease onset and stages of progression. It has become apparent that ALS/FTD-causative genes, such as C9ORF72, may have roles in maintaining the normal physiology of the synapse, as mutations in these genes often manifest in synaptic dysfunction. Here we review the dysfunctions of the central nervous system synapses associated with the nucleotide repeat expansion in C9ORF72 observed in patients, organismal, and cellular models of ALS and FTD.

The effects of molecular crowding and CpG hypermethylation on DNA G-quadruplexes formed by the C9orf72 nucleotide repeat expansion.

A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf72 (C9), is the most common genetic mutation associated with ALS/FTD. There is increasing evidence that nucleic acid structures formed by the C9-NRE may both contribute to ALS/FTD, and serve as therapeutic targets, but there is limited characterization of these nucleic acid structures under physiologically and disease relevant conditions. Here we show in vitro that the C9-NRE DNA can form both parallel and antiparallel DNA G-quadruplex (GQ) topological structures and that the structural preference of these DNA GQs can be dependent on the molecular crowding conditions. Additionally, 5-methylcytosine DNA hypermethylation, which is observed in the C9-NRE locus in some patients, has minimal effects on GQ topological preferences. Finally, molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions. These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases.

C9orf72 deficiency promotes microglial-mediated synaptic loss in aging and amyloid accumulation.

C9orf72 repeat expansions cause inherited amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) and result in both loss of C9orf72 protein expression and production of potentially toxic RNA and dipeptide repeat proteins. In addition to ALS/FTD, C9orf72 repeat expansions have been reported in a broad array of neurodegenerative syndromes, including Alzheimer's disease. Here we show that C9orf72 deficiency promotes a change in the homeostatic signature in microglia and a transition to an inflammatory state characterized by an enhanced type I IFN signature. Furthermore, C9orf72-depleted microglia trigger age-dependent neuronal defects, in particular enhanced cortical synaptic pruning, leading to altered learning and memory behaviors in mice. Interestingly, C9orf72-deficient microglia promote enhanced synapse loss and neuronal deficits in a mouse model of amyloid accumulation while paradoxically improving plaque clearance. These findings suggest that altered microglial function due to decreased C9orf72 expression directly contributes to neurodegeneration in repeat expansion carriers independent of gain-of-function toxicities.

Evaluation of the Acute Flaccid Paralysis Surveillance System in Polio-Free Jordan, 2012-2016: Retrospective Secondary Analysis.

BACKGROUND: As part of the polio-eradication strategy, the World Health Organization (WHO) has established a global acute flaccid paralysis (AFP) surveillance system. AFP surveillance has successfully helped Jordan achieve polio-free certification. However, there is a substantial risk of polio importation from neighboring countries including Syria and Iraq. OBJECTIVE: This study aimed to evaluate the AFP surveillance in Jordan and identify areas that need improvement. METHODS: This retrospective study is a secondary analysis of data that were routinely collected between 2012 and 2016 by Jordan's Expanded Program on Immunization. The WHO's minimum performance indicators were used to evaluate the AFP surveillance. RESULTS: Cumulatively, 328 AFP cases had been reported. Almost half (n=168, 51.3%) of the patients were aged 1-5 years, and 55.8% (n=183) were male. All cases were discarded (classified as a nonpolio case). The most common cause of AFP was Guillain-Barre Syndrome (n=115, 35.1%). The annualized nonpolio AFP rate increased from 1.4/100,000 children below 15 years of age in 2012 to 4.3 in 2016. National and subnational sensitivities were not met in 2012 and 2013. Adequacy of stool specimens and timeliness of specimens arriving at and processed in the laboratory were constantly above the minimum target. Timeliness of the investigation met the expected target but with a decreasing trend. The nonpolio enterovirus isolation rate was below the target, except in 2016. CONCLUSIONS: The AFP surveillance system in Jordan is performing well; however, additional efforts are needed to strengthen the subnational sensitivity. The cold chain from sample collection to laboratory testing has to be maintained to ensure the reliability of stool specimens required for isolation of the nonpolio enterovirus.

Representing Diversity in the Dish: Using Patient-Derived in Vitro Models to Recreate the Heterogeneity of Neurological Disease.

Neurological diseases, including dementias such as Alzheimer's disease (AD) and fronto-temporal dementia (FTD) and degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS), are responsible for an increasing fraction of worldwide fatalities. Researching these heterogeneous diseases requires models that endogenously express the full array of genetic and epigenetic factors which may influence disease development in both familial and sporadic patients. Here, we discuss the two primary methods of developing patient-derived neurons and glia to model neurodegenerative disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which are differentiated into neurons or glial cells, or directly converting (DC) somatic cells into neurons (iNeurons) or glial cells. Distinct differentiation techniques for both models result in a variety of neuronal and glial cell types, which have been successful in displaying unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease of genetic manipulation, expression of cell-specific markers, and recapitulation of disease pathogenesis are presented as determining factors in how these methods may be used separately or together to ascertain mechanisms of disease and identify therapeutics for distinct patient populations or for specific individuals in personalized medicine projects.

Comparison of Analysis Tools for miRNA High Throughput Sequencing Using Nerve Crush as a Model.

Recent advances in sample preparation and analysis for next generation sequencing have made it possible to profile and discover new miRNAs in a high throughput manner. In the case of neurological disease and injury, these types of experiments have been more limited. Possibly because tissues such as the brain and spinal cord are inaccessible for direct sampling in living patients, and indirect sampling of blood and cerebrospinal fluid are affected by low amounts of RNA. We used a mouse model to examine changes in miRNA expression in response to acute nerve crush. We assayed miRNA from both muscle tissue and blood plasma. We examined how the depth of coverage (the number of mapped reads) changed the number of detectable miRNAs in each sample type. We also found that samples with very low starting amounts of RNA (mouse plasma) made high depth of mature miRNA coverage more difficult to obtain. Each tissue must be assessed independently for the depth of coverage required to adequately power detection of differential expression, weighed against the cost of sequencing that sample to the adequate depth. We explored the changes in total mapped reads and differential expression results generated by three different software packages: miRDeep2, miRNAKey, and miRExpress and two different analysis packages, DESeq and EdgeR. We also examine the accuracy of using miRDeep2 to predict novel miRNAs and subsequently detect them in the samples using qRT-PCR.