Quantification of purified endogenous miRNAs with high sensitivity and specificity

AbstractMicroRNAs (miRNAs) are short (19–24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3′-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.

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Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA

Communications Biology ◽

10.1038/s42003-021-01648-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Zhen Wang ◽

Jérôme Maluenda ◽

Laurène Giraut ◽

Thibault Vieille ◽

Andréas Lefevre ◽

...

Keyword(s):

Single Molecule ◽

Fragile X ◽

Methylation Status ◽

Single Molecules ◽

High Sensitivity ◽

E Coli ◽

Dna And Rna ◽

Magnetic Tweezer ◽

Base Modifications ◽

Sensitivity Specificity

AbstractAccurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5’UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.

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Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

10.1101/2020.04.03.002501 ◽

2020 ◽

Author(s):

Zhen Wang ◽

Jérôme Maluenda ◽

Laurène Giraut ◽

Thibault Vieille ◽

Andréas Lefevre ◽

...

Keyword(s):

Single Molecule ◽

Fragile X ◽

Methylation Status ◽

Single Molecules ◽

High Sensitivity ◽

Base Modification ◽

Dna And Rna ◽

Magnetic Tweezer ◽

Base Modifications ◽

Sensitivity Specificity

AbstractAccurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5‘UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

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TO SEPARATELY ASSESS THE DIAGNOSTIC POTENTIAL OF TVS & MRI IN THE TERMS OF SENSITIVITY AND SPECIFICITY BY CORRELATING THE IMAGING FINDINGS WITH HISTOPATHOLOGY

International Journal of Medical and Biomedical Studies ◽

10.32553/ijmbs.v4i6.1209 ◽

2020 ◽

Vol 4 (6) ◽

Author(s):

Suraj Mathur

Keyword(s):

Transvaginal Ultrasound ◽

High Sensitivity ◽

Imaging Evaluation ◽

Medical College ◽

Conventional Mri ◽

Diagnostic Potential ◽

Adc Mapping ◽

Malignant Lesions ◽

Sensitivity Specificity

This prospective study was done in the Department of Radio diagnosis Govt. Medical College, Kozhikode. A total of 65 patients who were referred to our department with clinical suspicion of endometrial lesions and incidentally detected endometrial lesions on ultrasonography underwent transvaginal ultrasound and subsequent Imaging evaluation of pelvis MRI has very high sensitivity (95%) and specificity (98%) and is almost as accurate (97%) as histopathology in differentiating benign from malignant lesions. Addition of DWI with ADC mapping to conventional MRI increases its accuracy even more. However there is inherent limitation to MRI in detecting carcinoma in situ and micrometastasis. Keywords: TVS, MRI, Sensitivity, Specificity, Histopathology.

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Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

10.26434/chemrxiv.10080638.v2 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Meng-Yin Li ◽

Jie Yang ◽

Ya-Qian Wang ◽

Xue-Yuan Wu ◽

...

Keyword(s):

Dna Sequencing ◽

Single Molecule ◽

Genetic Diseases ◽

High Sensitivity ◽

Dna Lesions ◽

Lesion Detection ◽

The Novel ◽

Structural Variations ◽

Dna Lesion ◽

Base Lesion

DNA lesion such as metholcytosine(mC), 8-OXO-guanine(OG), inosine(I) etc could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (mC, OG, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

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UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200424130934 ◽

2020 ◽

Vol 20 (12) ◽

pp. 1487-1496 ◽

Cited By ~ 1

Author(s):

Midori Murakami ◽

Hiroto Izumi ◽

Tomoko Kurita ◽

Chiho Koi ◽

Yasuo Morimoto ◽

...

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Anticancer Agent ◽

Cisplatin Resistance ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Molecular Target ◽

Transcriptional Level ◽

And Control ◽

Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

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Diploid chromosome-scale assembly of the Muscadinia rotundifolia genome supports chromosome fusion and disease resistance gene expansion during Vitis and Muscadinia divergence

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab033 ◽

2021 ◽

Author(s):

Noé Cochetel ◽

Andrea Minio ◽

Mélanie Massonnet ◽

Amanda M Vondras ◽

Rosa Figueroa-Balderas ◽

...

Keyword(s):

Single Molecule ◽

Interleukin 1 ◽

Plasmopara Viticola ◽

Cabernet Sauvignon ◽

Disease Resistance Gene ◽

Chromosome Fusion ◽

Protein Coding ◽

Downy Mildews ◽

A Genome ◽

Muscadinia Rotundifolia

Abstract Muscadinia rotundifolia, the muscadine grape, has been cultivated for centuries in the southeastern United States. M. rotundifolia is resistant to many of the pathogens that detrimentally affect Vitis vinifera, the grape species commonly used for winemaking. For this reason, M. rotundifolia is a valuable genetic resource for breeding. Single-molecule real-time reads were combined with optical maps to reconstruct the two haplotypes of each of the 20 M. rotundifolia cv. Trayshed chromosomes. The completeness and accuracy of the assembly were confirmed using a high-density linkage map of M. rotundifolia. Protein-coding genes were annotated using an integrated and comprehensive approach. This included using Full-length cDNA sequencing (Iso-Seq) to improve gene structure and hypothetical spliced variant predictions. Our data strongly support that Muscadinia chromosomes 7 and 20 are fused in Vitis and pinpoint the location of the fusion in Cabernet Sauvignon and PN40024 chromosome 7. Disease-related gene numbers in Trayshed and Cabernet Sauvignon were similar, but their clustering locations were different. A dramatic expansion of the Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) class was detected on Trayshed chromosome 12 at the Resistance to Uncinula necator 1 (RUN1)/ Resistance to Plasmopara viticola 1 (RPV1) locus, which confers strong dominant resistance to powdery and downy mildews. A genome browser for Trayshed, its annotation, and an associated Blast tool are available at .www.grapegenomics.com

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Use of β-D-glucan in diagnosis of suspected Pneumocystis jirovecii pneumonia in adults with HIV infection

International Journal of STD & AIDS ◽

10.1177/09564624211022247 ◽

2021 ◽

pp. 095646242110222

Author(s):

Thomas Juniper ◽

Chris P Eades ◽

Eliza Gil ◽

Harriet Fodder ◽

Killian Quinn ◽

...

Keyword(s):

Predictive Value ◽

Diagnostic Tests ◽

Elevated Serum ◽

Clinical Information ◽

High Sensitivity ◽

Pneumocystis Pneumonia ◽

Pneumocystis Jirovecii Pneumonia ◽

Alternative Diagnosis ◽

Positive Threshold ◽

Sensitivity Specificity

Objectives: An elevated serum (1-3)-β-D-glucan (BDG) concentration has high sensitivity for a diagnosis of Pneumocystis pneumonia (PCP) in people with HIV (PWH). At the current manufacturer-recommended positive threshold of 80 pg/mL (Fungitell), specificity for PCP is variable and other diagnostic tests are required. We evaluated the utility of serum BDG for diagnosis of suspected PCP in PWH at three inner-London hospitals to determine BDG concentrations for diagnosis and exclusion of PCP. Methods: From clinical case records, we abstracted demographic and clinical information and categorised patients as having confirmed or probable PCP, or an alternative diagnosis. We calculated sensitivity, specificity and positive predictive value (PPV) of serum BDG concentrations >400 pg/mL and negative predictive value (NPV) of BDG <80 pg/mL. Results: 76 patients were included; 29 had laboratory-confirmed PCP, 17 had probable PCP and 30 had an alternative diagnosis. Serum BDG >400 pg/mL had a sensitivity of 83%, specificity of 97% and PPV 97% for diagnosis of PCP; BDG <80 pg/mL had 100% NPV for exclusion of PCP. Conclusions: In PWH with suspected PCP, BDG <80 pg/mL excludes a diagnosis of PCP, whereas BDG concentrations >400 pg/mL effectively confirm the diagnosis. Values 80–400 pg/mL should prompt additional diagnostic tests.

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The Child Behavior Checklist as a Screening Instrument for PTSD in Refugee Children

Children ◽

10.3390/children8060521 ◽

2021 ◽

Vol 8 (6) ◽

pp. 521

Author(s):

Ina Nehring ◽

Heribert Sattel ◽

Maesa Al-Hallak ◽

Martin Sack ◽

Peter Henningsen ◽

...

Keyword(s):

Child Behavior ◽

Child Behavior Checklist ◽

High Sensitivity ◽

Roc Curves ◽

Structured Interview ◽

Screening Instrument ◽

Screening Tools ◽

Refugee Children ◽

Post Traumatic Stress ◽

Sensitivity Specificity

Thousands of refugees who have entered Europe experienced threatening conditions, potentially leading to post traumatic stress disorder (PTSD), which has to be detected and treated early to avoid chronic manifestation, especially in children. We aimed to evaluate and test suitable screening tools to detect PTSD in children. Syrian refugee children aged 4–14 years were examined using the PTSD-semi-structured interview, the Kinder-DIPS, and the Child Behavior Checklist (CBCL). The latter was evaluated as a potential screening tool for PTSD using (i) the CBCL-PTSD subscale and (ii) an alternative subscale consisting of a psychometrically guided selection of items with an appropriate correlation to PTSD and a sufficient prevalence (presence in more than 20% of the cases with PTSD). For both tools we calculated sensitivity, specificity, and a receiver operating characteristic (ROC) curve. Depending on the sum score of the items, the 20-item CBCL-PTSD subscale as used in previous studies yielded a maximal sensitivity of 85% and specificity of 76%. The psychometrically guided item selection resulted in a sensitivity of 85% and a specificity of 83%. The areas under the ROC curves were the same for both tools (0.9). Both subscales may be suitable as screening instrument for PTSD in refugee children, as they reveal a high sensitivity and specificity.

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Evaluation of HiCrome KPC Agar for the Screening of Carbapenem-Resistant Enterobacterales Colonization in the ICU Setting of a Tertiary Care Hospital

Journal of Laboratory Physicians ◽

10.1055/s-0041-1732494 ◽

2021 ◽

Author(s):

Ashoka Mahapatra ◽

K Nikitha ◽

Sutapa Rath ◽

Bijayini Behera ◽

Kavita Gupta

Keyword(s):

Tertiary Care ◽

High Sensitivity ◽

Care Hospital ◽

Predictive Values ◽

Klebsiella Pneumoniae Carbapenemase ◽

Carbapenem Resistant ◽

Rectal Swabs ◽

Control And Prevention ◽

Indian Setting ◽

Sensitivity Specificity

Abstract Background Spread of carbapenem-resistant Enterobacterales (CRE) is a significant concern in intensive care unit (ICU) settings. Approaches to routine screening for CRE colonization in all ICU patients vary depending on institutional epidemiology and resources. The present study was aimed to evaluate the performance of HiCrome Klebsiella pneumoniae carbapenemase (KPC) agar for the detection of CRE colonization in ICU settings taking the Centers for Disease Control and Prevention (CDC) recommended method as reference. Methods Two-hundred and eighty rectal swabs (duplicate) from 140 patients were subjected to CRE detection in HiCrome KPC agar and MacConkey agar (CDC criteria). Results Using CDC method, total 41 CRE isolates were recovered comprising of 29 E scherichia coli, 11 Klebsiella, and 1 Enterobacter spp. On the other hand, 49 isolates of CRE recovered from 140 rectal swabs using HiCrome KPC agar, out of which 33 were E. coli, 15 Klebsiella, and 1 Enterobacter sp. Statistical Analysis Sensitivity, specificity, negative, and positive predictive values of CRE screening by HiCrome KPC agar were found to be 100% (91.4–100), 91.9% (84.8–95.8), 83.6% (70.9–91.4), and 100% (95.9–100), respectively, taking the CDC recommended method as reference. Conclusion HiCrome KPC agar has high sensitivity in screening CRE colonization. Further studies are needed to establish its applicability for detecting the predominant circulating carbapenemases in the Indian setting.

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4 Molecularly guided multiplexed digital spatial analysis reveals differential gene expression profiles in the WNT-β-catenin pathway between melanoma and prostate tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0004 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A4-A4

Author(s):

Anushka Dikshit ◽

Dan Zollinger ◽

Karen Nguyen ◽

Jill McKay-Fleisch ◽

Kit Fuhrman ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Molecule ◽

Spatial Information ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Fluorescence Assay ◽

Differentially Expressed ◽

Tumor Type ◽

Prostate Tumors

BackgroundThe canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression.MethodsAfter screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution.ResultsThe GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway.ConclusionsIn summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.

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