Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry.

Precise determination of ribonucleic acid (RNA) concentration without the need for calibration was pursued by sequence-specific counting of individual RNA molecules. This approach eliminates the reverse transcription (RT) step required for polymerase chain reaction (PCR)-based quantification, which may hamper accurate measurements owing to uncertain yields of RT reactions. Target RNAs were tagged with a number of fluorescent oligonucleotide probes with complementary sequences. Tagged RNAs were exhaustively counted one by one using a high-sensitivity capillary-based flow cytometric setup. MS2 viral RNA was quantified as a model RNA for which essential parameters, including probe numbers, probe concentration, and hybridization conditions, were optimized for the best performance. Using 70 oligonucleotide probes, MS2 RNA was quantified with 2.0% relative standard deviation, and its validity was assessed by comparison with other RNA quantification methods such as droplet digital PCR and UV spectrophotometry. The observed comparability indicated that the proposed method is unlikely to have a substantial bias. It works for a substantially lower-level RNA than UV and avoids the potential variability in the yield of the RT reaction of RT-qPCR. Therefore, the proposed method could be a valuable addition to current methods and could be further developed as a standard reference method for RNA quantification.

High-sensitivity microvolume UV absorption spectrometry for routine analysis of small-volume biological samples.

Substantial improvement of microvolume UV absorption spectrometry in sensitivity, robustness and ease of operation was achieved for routine biological applications. A unique microtubing-based absorption cell (208 µm internal diameter) featuring enhanced light transmission with a liquid core waveguide technique provided dramatically enhanced absorption sensitivity, proportional to the extended path length (50 mm, from the typical 1 mm), while robust measurement performance was attained by implementation of preventive measures against bubble trapping along the light path. For pBR322 plasmid DNA, absorbance at 260 nm was reliably measurable down to 0.1 ng/µl with repeatability typically 2-3% RSD. The detection limit was 0.03 ng/µl dsDNA, far lower than the current state-of-the-art âˆ¼1 ng/µl. Sample consumption for each measurement was 2.4 µl. Automated operation implemented for the first time in microvolume spectrophotometry facilitated the ease in handling with small-volume biological samples.

A High Quality Asian Genome Assembly Identifies Features of Common Missing Regions.

The current human reference genome (GRCh38), with its superior quality, has contributed significantly to genome analysis. However, GRCh38 may still underrepresent the ethnic genome, specifically for Asians, though exactly what we are missing is still elusive. Here, we juxtaposed GRCh38 with a high-contiguity genome assembly of one Korean (AK1) to show that a part of AK1 genome is missing in GRCh38 and that the missing regions harbored ~1390 putative coding elements. Furthermore, we found that multiple populations shared some certain parts in the missing genome when we analyzed the "unmapped" (to GRCh38) reads of fourteen individuals (five East-Asians, four Europeans, and five Africans), amounting to ~5.3 Mb (~0.2% of AK1) of the total genomic regions. The recovered AK1 regions from the "unmapped reads", which were the estimated missing regions that did not exist in GRCh38, harbored candidate coding elements. We verified that most of the common (shared by ≥7 individuals) missing regions exist in human and chimpanzee DNA. Moreover, we further identified the occurrence mechanism and ethnic heterogeneity as well as the presence of the common missing regions. This study illuminates a potential advantage of using a pangenome reference and brings up the need for further investigations on the various features of regions globally missed in GRCh38.

A Deep Learning Approach for Estimating Traffic Density Using Data Obtained from Connected and Autonomous Probes.

The focus of this research is on the estimation of traffic density from data obtained from Connected and Autonomous Probes (CAPs). CAPs pose an advantage over expensive and invasive infrastructure such as loop detectors. CAPs maneuver their driving trajectories, sensing the presence of adjacent vehicles and distances to them by means of several electronic sensors, whose data can be used for more sophisticated traffic density estimation techniques. Traffic density has a highly nonlinear nature during on-congestion and queue-clearing conditions. Closed-mathematical forms of the traditional density estimation techniques are incapable of dealing with complex nonlinearities, which opens the door for data-driven approaches such as machine learning techniques. Deep learning algorithms excel in data-rich contexts, which recognize nonlinear and highly situation-dependent patterns. Our research is based on an LSTM (Long short-term memory) neural network for the nonlinearity associated with time dynamics of traffic flow. The proposed method is designed to learn the input-output relation of Edie's definition. At the same time, the method recognizes a temporally nonlinear pattern of traffic. We evaluate our algorithm by using a microscopic simulation program (PARAMICS) and demonstrate that our model accurately estimates traffic density in Free-flow, Transition, and Congested conditions.

Staged, Combined Management of Ruptured Vertebral Artery Dissecting Aneurysms Involving the Posterior Inferior Cerebellar Artery: Report of 4 Cases and Review of the Literature.

BACKGROUND: Ruptured vertebral artery dissecting aneurysms (VADAs) involving the posterior inferior cerebellar artery (PICA) are the most difficult to treat among variations of VADAs but require prompt treatment. The major challenge is to preserve the PICA while occluding the aneurysm. Despite advances in the management of ruptured VADAs involving the PICA, each treatment, whether it is combined or not, is associated with a significant degree of risk. CASE DESCRIPTION: This study presents 4 cases of ruptured VADAs involving the PICA that were successfully treated using a staged, combined method. Embolization of the rupture point in the acute stage was followed 3-4 weeks later by occlusion of the proximal vertebral artery (VA) and PICA origin after occipital artery-PICA bypass in the chronic stage. CONCLUSIONS: Although it is sometimes very difficult to determine the exact rupture point of VADAs, ruptures tend to occur at distal segments of a dissecting aneurysm presenting as bleb. Also, when planning a trapping of the VA, careful examination of angiography is needed to assess the contralateral VA and rupture point. Our staged and combined strategy may provide another valuable treatment option for treating VADAs involving the PICA with special emphasis on the safety and efficacy in our method.

Bifrontal Interhemispheric Approach Involving Cutting the Superior Sagittal Sinus for Distal Anterior Cerebral Artery Aneurysms.

BACKGROUND: The unilateral interhemispheric approach for distal anterior cerebral artery aneurysms presents several risks, such as postoperative venous infarction due to occasional sacrifice of parasagittal bridging vein and postoperative frontal lobe damage due to retraction force. To overcome these risks, we used a bifrontal craniotomy with straight dural incision and cutting of the superior sagittal sinus. METHODS: We retrospectively reviewed 61 patients (42 unruptured and 19 ruptured A2 and A3 aneurysms) who under aneurysm clipping through bifrontal interhemispheric approach between March 2007 and December 2017. There were 35 A2 aneurysms and 27 A3 aneurysms, and mean size of aneurysms was 5.45 mm. The modified bifrontal interhemispheric approach involved 3 steps: bifrontal craniotomy of centrobasal portion of the frontal bone, ligation and division of anterior one third of the superior sagittal sinus, and approaching the aneurysm via the interhemispheric space. All patients underwent computed tomography on postoperative days 3 and 7 for evaluation of brain retraction damage or venous infarction. RESULTS: Among patients with ruptured aneurysms, 79% had a favorable outcome (Glasgow Outcome Scale score 4 or 5) 6 months after primary subarachnoid hemorrhage; all patients with unruptured aneurysms had favorable outcomes. Surgical outcome was strongly related to preoperative neurologic Hunt and Hess grade. Three patients had poor outcomes due to poor Hunt and Hess grade on admission (grade 3 in 2 patients, grade 4 in 1 patient). Follow-up CT showed that venous infarction did not occur in any of the patients. CONCLUSIONS: Modified bifrontal interhemispheric approach may be a safe and effective method for treating A2 and A3 aneurysms with relatively good clinical outcome and no surgery-related complications.

Stable Isotope Labeled DNA: A New Strategy for the Quantification of Total DNA Using Liquid Chromatography-Mass Spectrometry.

Conventional DNA quantification methods require a DNA purification step that limits their reliability in estimating the original DNA amount, especially in complex matrix. To overcome this limitation, we developed a method to calibrate the variable DNA extraction efficiencies during the purification process, allowing for the accurate quantification of DNA in complex matrix. This method is based on isotope dilution-liquid chromatography-mass spectrometry using stable isotope labeled DNA (SILD) as an internal standard. Steps include spiking prepared SILD into samples, purification, enzymatic hydrolysis, and detection of DNA monomers via mass spectrometry, where the spiked SILD is expected to behave the same as the target DNA throughout the entire procedure. We show that the mean recoveries of four different DNA purification kits were dramatically improved by using the SILD internal standard, both for Escherichia coli and human genomic DNA. As standards for calibration, deoxyribonucleoside monophosphates and purified genomic DNA were tested, with genomic DNA from corresponding species found to calibrate the variable extraction efficiencies more effectively. With this successful calibration, our newly developed procedure enables International System of Units-traceable quantification of total DNA in complex matrix.

Real-Time Light-Guided Vocal Fold Injection: Ex Vivo Feasibility Study in a Canine Model.

OBJECTIVE: The transcricothyroid (CT) membrane approach is a good option for office-based vocal fold injection (VFI). However, because the needle tip is invisible during injection using the CT approach, precise localization requires a high level of experience, and mastering this approach involves a steep learning curve. To overcome current limitations, we conceptualized a novel technique: real-time light-guided VFI (RL-VFI), which enables simultaneous VFI under direct visualization of the lighted needle tip. Herein, we aimed to verify the feasibility of RL-VFI in cadaveric canine model, simulating the setting of office-based VFI, as well as to explore its clinical usefulness. STUDY DESIGN: Animal study. METHODS: A customized prototype device was developed. It consisted of three parts: light source, controller, and injector. Light source comprised laser diodes of two wavelengths (635 nanometers [nm], red; 532 nm, green). Four types of injector were developed using 40-mm needles of 23- and 25-gauge and optic fibers of 50 and 100 µm. ex vivo canine larynx was prepared for the experiment. Flexible laryngoscopy system was used to examine canine vocal folds. RESULTS: Various routes from three insertion points (3 mm, 10 mm, and 17 mm from the midline) were validated using the device. Regardless of the injection routes, the location of the needle tip was accurately indicated by light. RL-VFI was feasible under light guidance without difficulties. Moreover, precise and simultaneous re-injection could be performed at the intended point using the device. CONCLUSION: We introduced RL-VFI using our customized prototype device in an ex vivo canine larynx, simulating the setting of office-based VFI. Clinical application of RL-VFI will improve safety and precision of CT approach, as well as expand its applications in laryngology. LEVEL OF EVIDENCE: NA. Laryngoscope, 129:935-942, 2019.

Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard.

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs, the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach, as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition, incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results, we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol, which will improve both accuracy and comparability of NGS results across different sample batches, laboratories and NGS platforms.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

Effect of Device Rigidity and Physiological Loading on Spinal Kinematics after Dynamic Stabilization : An In-Vitro Biomechanical Study.

OBJECTIVE: To investigate the effects of posterior implant rigidity on spinal kinematics at adjacent levels by utilizing a cadaveric spine model with simulated physiological loading. METHODS: Five human lumbar spinal specimens (L3 to S1) were obtained and checked for abnormalities. The fresh specimens were stripped of muscle tissue, with care taken to preserve the spinal ligaments and facet joints. Pedicle screws were implanted in the L4 and L5 vertebrae of each specimen. Specimens were tested under 0 N and 400 N axial loading. Five different posterior rods of various elastic moduli (intact, rubber, low-density polyethylene, aluminum, and titanium) were tested. Segmental range of motion (ROM), center of rotation (COR) and intervertebral disc pressure were investigated. RESULTS: As the rigidity of the posterior rods increased, both the segmental ROM and disc pressure at L4-5 decreased, while those values increased at adjacent levels. Implant stiffness saturation was evident, as the ROM and disc pressure were only marginally increased beyond an implant stiffness of aluminum. Since the disc pressures of adjacent levels were increased by the axial loading, it was shown that the rigidity of the implants influenced the load sharing between the implant and the spinal column. The segmental CORs at the adjacent disc levels translated anteriorly and inferiorly as rigidity of the device increased. CONCLUSION: These biomechanical findings indicate that the rigidity of the dynamic stabilization implant and physiological loading play significant roles on spinal kinematics at adjacent disc levels, and will aid in further device development.

Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

SiNG-PCRseq: Accurate inter-sequence quantification achieved by spiking-in a neighbor genome for competitive PCR amplicon sequencing.

Despite the recent technological advances in DNA quantitation by sequencing, accurate delineation of the quantitative relationship among different DNA sequences is yet to be elaborated due to difficulties in correcting the sequence-specific quantitation biases. We here developed a novel DNA quantitation method via spiking-in a neighbor genome for competitive PCR amplicon sequencing (SiNG-PCRseq). This method utilizes genome-wide chemically equivalent but easily discriminable homologous sequences with a known copy arrangement in the neighbor genome. By comparing the amounts of selected human DNA sequences simultaneously to those of matched sequences in the orangutan genome, we could accurately draw the quantitative relationships for those sequences in the human genome (root-mean-square deviations <0.05). Technical replications of cDNA quantitation performed using different reagents at different time points also resulted in excellent correlations (R(2) > 0.95). The cDNA quantitation using SiNG-PCRseq was highly concordant with the RNA-seq-derived version in inter-sample comparisons (R(2) = 0.88), but relatively discordant in inter-sequence quantitation (R(2) < 0.44), indicating considerable level of sequence-dependent quantitative biases in RNA-seq. Considering the measurement structure explicitly relating the amount of different sequences within a sample, SiNG-PCRseq will facilitate sharing and comparing the quantitation data generated under different spatio-temporal settings.

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry.

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and (31)P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).

Quantification of trace-level DNA by real-time whole genome amplification.

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive.

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2'-deoxyribonucleoside 5'-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.

The anti-aging gene KLOTHO is a novel target for epigenetic silencing in human cervical carcinoma.

BACKGROUND: Klotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. In this study, we examined epigenetic silencing of KLOTHO in human cervical carcinoma. RESULTS: Loss of KLOTHO mRNA was observed in several cervical cancer cell lines and in invasive carcinoma samples, but not during the early, preinvasive phase of primary cervical tumorigenesis. KLOTHO mRNA was restored after treatment with either the DNA demethylating agent 2'-deoxy-5-azacytidine or histone deacetylase inhibitor trichostatin A. Methylation-specific PCR and bisulfite genomic sequencing analysis of the promoter region of KLOTHO revealed CpG hypermethylation in non-KLOTHO-expressing cervical cancer cell lines and in 41% (9/22) of invasive carcinoma cases. Histone deacetylation was also found to be the major epigenetic silencing mechanism for KLOTHO in the SiHa cell line. Ectopic expression of the secreted form of KLOTHO restored anti-Wnt signaling and anti-clonogenic activity in the CaSki cell line including decreased active beta-catenin levels, suppression of T-cell factor/beta-catenin target genes, such as c-MYC and CCND1, and inhibition of colony growth. CONCLUSIONS: Epigenetic silencing of KLOTHO may occur during the late phase of cervical tumorigenesis, and consequent functional loss of KLOTHO as the secreted Wnt antagonist may contribute to aberrant activation of the canonical Wnt pathway in cervical carcinoma.

p15INK4b methylation correlates with thrombocytopenia, blast percentage, and survival in myelodysplastic syndromes in a dose dependent manner: quantitation using pyrosequencing study.

We investigated how the quantity of p15INK4b methylation related to International Prognosic Scoring System variables and survival in 74 patients with de novo myelodysplastic syndrome (MDS). Pyrosequencing of 11 consecutive CpG sites of the p15INK4b promotor region was performed, with the extent of CpG cytosine methylation assessed in terms of methylation level (MtL). Patients with >5% bone marrow blasts had higher MtL than patients with <5% blasts (10.1% vs. 6.1%, p=0.030, respectively). Methylation was not associated with chromosomal aberrations. The MtL of patients with thrombocytopenia were higher than patients without thrombocytopenia (11.2% vs. 6.2%, p=0.036, respectively); they were higher in patients with cytopenias in > or =2 lineages than in patients with either unilineage or no cytopenia (9.8% vs. 4.1%, p=0.036, respectively). The survival of patients with >7% MtL was worse than patients with <7% MtL (p=0.031). Heavy p15INK4b methylation in MDS is associated with IPSS predictors of poor prognosis and adverse survival.

An international comparability study on quantification of total methyl cytosine content.

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.