Assessing the Utility of Thermodynamic Features for microRNA Target Prediction under Relaxed Seed and No Conservation Requirements

BACKGROUND: Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5'seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement. METHODOLOGY/PRINCIPAL FINDINGS: We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3-5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules

Raman spectroscopy: techniques and applications in the life sciences

Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine. It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration

Investigations of the supramolecular structure of individual diphenylalanine nano- and microtubes by polarised Raman microspectroscopy

This work presents the development of an integrated instrument which incorporates two powerful techniques , the atomic force microscopy (AFM) and the polarised Raman micro-spectroscopy (PR~!lS), to determine the molecular organisation of the Phe-Phe (FF) micro- and nanotubes. The diphenylalanine peptidebased nanotubes consist of the FF molecules self-aggregated into a tubular structure exhibiting unique physical, chemical and biological properties. These prominent characteristics promote this nanomaterial to be a potential candidate for numerous applications. The FF peptide-based structure has also been studied extensively in medical research. This is because its self-aggregation mechanism plays a key role in the formation of amyloid fibrils , which are related to various diseases. Understanding such formation mechanism could provide new treatments for these diseases. The structural organisation of the FF tubes became a controversial issue because of the lack of direct evidence, especially at micro- and nanoscale. In this ~work , AFM and polarised Raman spectroscopy was used to determine molecular interaction and orientation for individual FF tubes. As the combined AFM-PRMS system consists of two essential parts operating individually, new control software was designed and developed to synchronise both instruments. Therefore, investigation of the selected FF tubes can be performed using polarised Raman micro-spect;~scopy. Simultaneously, the tube diameter can be measured accurately using the AFM. To determine the molecular organisation of the FF tubes , the studied began with the investigation of the FF samples by conventional Raman measurements for determining the observed Raman bands and their corresponding vibrational modes. Additionally, the DFT calculation was employed to confirm the band assignment. Finally, the polarised Raman micro-spectroscopy was used to determined the anisotropic properties of some key vibrational modes, particularly, the amide I and the amide III bands. The experimental measurements were carried out by two methods. In the first method. the FF samples was studied by investigating the variation of intensity of the selected Raman bands when the polarisation direction of excitation laser changed from 0° to 360° vvith respect to the tube axis. The probable orientation of the selected bands was depicted in polar diagrams of normalised Raman intensity. For the second approach, the FF tubes were investigated with four polarisation configurations using a half-wave plate and an analyser. A dataset of polarised Raman spectra corresponding to the selected bands was mathematically analysed using a uniaxial model to determine quantitatively the orientation distribution function (ODF). Both methods provided consistent results illustrating that the peptide C=O bonds of the FF molecule are preferentially aligned along the tube and the peptide backbone is aligned perpendicular of the tube. From the second method , the angle between the C=O bond and the tube axis was determined to be rv 8° in crystalline structure. Furthermore, the tubes of different diameter showed no noticeable variation in molecular organisation within the experimental error. As the calculation was dependent on various parameters, the uncertainty of the mean orientation angle was also assessed. IIEThOS - Electronic Theses Online ServiceGBUnited Kingdo

MicroRNA Target Prediction via Duplex Formation Features and Direct Binding Evidence

<p>MicroRNAs (miRNAs) are small RNAs that have important roles in post-transcriptional gene regulation in a wide range of species. This regulation is controlled by having miRNAs directly bind to a target messenger RNA (mRNA), causing it to be destabilized and degraded, or translationally repressed. Identifying miRNA targets has been a large area of focus for study; however, a lack of generally high-throughput experiments to validate direct miRNA targeting has been a limiting factor. To overcome these limitations, computational methods have become crucial for understanding and predicting miRNA-gene target interactions.</p><p>While a variety of computational tools exist for predicting miRNA targets, many of them are focused on a similar feature set for their prediction. These commonly used features are complementarity to 5'seed of miRNAs and evolutionary conservation. Unfortunately, not all miRNA target sites are conserved or adhere to canonical seed complementarity. Seeking to address these limitations, several studies have included energy features of mRNA:miRNA duplex formation as alternative features. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement.</p><p>We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide interaction sites to Argonaute (AGO) protein family members, which are essential parts of the miRNA machinery complex. This trend is confirmed on data sets that assay the effect of miRNAs on mRNA and protein expression changes, where a statistically significant change in expression is noted when compared to the control. Furthermore, our method also allows for prediction of strictly imperfect sites, as well as non-conserved targets.</p><p>Recently, new methods for identifying direct miRNA binding have been developed, which provides us with additional sources of information for miRNA target prediction. While some computational target predictions tools have begun to incorporate this information, they still rely on the presence of a seed match in the AGO-bound windows without accounting for the possibility of variations. </p><p>We investigate the usefulness of the site level direct binding evidence in miRNA target identification and propose a model that incorporates multiple different features along with the AGO-interaction data. Our method outperforms both an ad hoc strategy of seed match searches as well as an existing target prediction tool, while still allowing for predictions of sites other than a long perfect seed match. Additionally, we show supporting evidence for a class of non-canonical sites as bound targets. Our model can be extended to predict additional types of imperfect sites, and can also be readily modified to include additional features that may produce additional improvements.</p>Dissertatio

Falsely Elevated 25-Hydroxy-Vitamin D Levels in Patients with Hypercalcemia

Symptomatic hypercalcemia is a commonly encountered clinical scenario. Though it is important to collect detailed history to find clinical clues connecting to the etiology of hypercalcemia, the diagnostic workup of hypercalcemia depends heavily on laboratory analysis. Accurate measurement of the parathyroid hormone and vitamin D levels is essential. However, commercial laboratory measurement of vitamin D levels can be erroneous in the setting of abundant paraprotein in the serum. One of the most common conditions that can cause an increased amount of paraproteins is multiple myeloma. We report 2 cases of falsely elevated 25-hydroxy-vitamin D levels in patients presenting with hypercalcemia and an underlying diagnosis of MM

Perioperative Intravenous Patient-Controlled Analgesic Efficacy of Morphine with Combined Nefopam and Parecoxib versus Parecoxib in Gynecologic Surgery: A Randomized, Double-Blind Study

Background. Nefopam is a non-NSAIDs and opioid sparing centrally acting drug which is effective for a multimodal postoperative analgesia. The present study aimed to evaluate the analgesic efficacy of nefopam combined with parecoxib for gynecologic surgery. Methods. This randomized double-blinded control trial recruited participants (n = 72) who underwent gynecologic surgeries and divided them into either a nefopam or control group. The study group received parecoxib 40 mg plus nefopam 20 mg, while the control group received parecoxib 40 mg plus normal saline solution intravenously during open abdominal gynecological surgery. Both groups then received either nefopam or normal saline every 6 hours postoperatively for 24 hours. Intravenous patient-controlled analgesia with morphine was given for breakthrough pain within 24 h. The participants were evaluated for morphine consumption within 24 hours and postoperative pain using a verbal numerical rating scale (VNRS) at a postanesthetic care unit, at 6-, 12-, and 24-hour postoperative periods. Adverse effects were recorded. Results. Morphine consumption within 24 hours and adverse effects were not significantly different between both groups. Mean difference and 95% confident interval of morphine consumption between both groups was 1.00 (−4.56, 4.76), P=0.97. The VNRS on movement at 6 hours after surgery of the nefopam group was significantly different from that of the control group [mean (SD), 4.14 (2.11) vs. 5.14 (1.80), P=0.04]. The VNRS of the nefopam group at 12 hours after operation during resting and on movement was significantly different from that of the control group ([mean (SD), 1.47 (1.80) vs. 2.54 (2.15), P=0.03], [mean (SD), 3.22 (1.84) vs 4.17 (1.74), P=0.03]), respectively. Conclusions. The combined administration of nefopam and parecoxib during gynecologic surgery slightly reduced the VNRS at 6 and 12 hours postoperatively more than treatment with parecoxib

Wettability gradient-induced alignment of peptide nanotubes as templates for biosensing applications

Self-assembled diphenylalanine (FF) peptide nanotubes (PNTs) have attracted significant attention due to their well-ordered supramolecular structure and wide range of functional capabilities that may enable potential nanobiotechnology applications. However, self-assembled PNTs are generally inhomogeneous at the macroscale, which has limited their potential use. Reproducibly controlling the assembly and alignment of PNTs is therefore critical to enable the widespread use of PNTs, e.g., in sensing applications. In this study, a surface patterning technique based on UV/ozone exposure through a mask is used to align PNTs. Exposed regions become hydrophilic, leading to directed spreading of the FF solution and alignment of the PNTs that improves as the difference in wettability between adjacent regions increases. Alignment was further found to depend on the concentration-and temperature-dependent diameter of the PNTs formed and the size of the hydrophilic area. Finally, aligned PNTs decorated with silver nanoparticles are used to sense an analyte molecule using surface enhanced Raman spectroscopy

MicroRNA target site identification by integrating sequence and binding information

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute proteins can pinpoint a microRNA (miRNA) target site within tens of bases but leaves the identity of the miRNA unresolved. A flexible computational framework, microMUMMIE, integrates sequence with cross-linking features and reliably identifies the miRNA family involved in each binding event. It considerably outperforms sequence-only approaches and quantifies the prevalence of noncanonical binding modes