Pratik Shah

Design aspects of bright red emissive silver nanoclusters/DNA probes for microRNA detection.

The influence of the nucleic acid secondary structure on the fast (1 h) formation of bright red emissive silver nanoclusters (AgNCs) in a DNA sequence (DNA-12nt-RED-160), designed for the detection of a microRNA sequence (RNA-miR160), was investigated. The findings show that especially the propensity for mismatch self-dimer formation of the DNA probes can be a good indicator for the creation and stabilization of red emissive AgNCs. Also, the role of the thermal stability of the secondary DNA structures (mismatch self-dimer and hairpin monomers) and the observed AgNC red emission intensity were investigated. These findings can form the basis for a rationale to design new red emissive AgNC-based probes. As an example, a bright red emissive AgNC-based DNA probe was designed for RNA-miR172 detection. The latter opens the possibility to create a variety of AgNC-based DNA probes for the specific detection of plant and animal miRNAs.

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes

MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method.

COP1 E3 ligase protects HYL1 to retain microRNA biogenesis

Constitutive photomorphogenic 1 (COP1) is a RING-finger E3 ligase that plays a central role in photomorphogenesis by destabilizing many light-regulated transcription factors and photoreceptors. Here, we reveal a novel function for COP1 E3 ligase in controlling global miRNA biogenesis in Arabidopsis thaliana. In cop1 mutants, the level of miRNAs is dramatically reduced because of the diminution of HYPONASTIC LEAVES 1 (HYL1), an RNA-binding protein required for precise miRNA processing. HYL1 is destabilized by an unidentified protease, which we tentatively call protease X, that specifically cleaves the N-terminal region from HYL1, thus neutralizing its function. Our results further show that the cytoplasmic partitioning of COP1 under light is essential to protect HYL1 against protease X. Taken together, we suggest a novel regulatory network involving HYL1, protease X, COP1 and light signalling that is indispensable for miRNA biogenesis in Arabidopsis thaliana.

Multiplexed microRNA Detection Using Lanthanide-Labeled DNA Probes and Laser Ablation Inductively Coupled Plasma Mass Spectrometry

In the past decade, microRNAs (miRNAs) have drawn increasing attention due to their role in regulation of gene expression. Especially, their potential as biomarkers in disease diagnostics has motivated miRNA research, including the development of simple, accurate, and sensitive detection methods. The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). Three miRNAs from Arabidopsis thaliana were analyzed simultaneously with high specificity, and the sensitivity of the method was comparable to radioactive detection (low femtomol range). The perspective of the developed method is highly multiplexed and quantitative miRNA analysis with high specificity and sensitivity.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes

MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

Post-Translational Regulation of miRNA Pathway Components, AGO1 and HYL1, in Plants.

Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis, the complexity of the proteome increases, and this then influences most biological processes. Although small RNAs are crucial regulatory elements for gene expression in most eukaryotes, PTMs of small RNA microprocessor and RNA silencing components have not been extensively investigated in plants. To date, several studies have shown that the proteolytic regulation of AGOs is important for host-pathogen interactions. DRB4 is regulated by the ubiquitin-proteasome system, and the degradation of HYL1 is modulated by a de-etiolation repressor, COP1, and an unknown cytoplasmic protease. Here, we discuss current findings on the PTMs of microprocessor and RNA silencing components in plants.

Incidence, microbiology, and outcomes of endophthalmitis after 111,876 pars plana vitrectomies at a single, tertiary eye care hospital

Purpose To describe the incidence, risk factors, clinical presentation, causative organisms, and outcomes in patients with endophthalmitis following pars plana vitrectomy (20G and minimally invasive vitrectomy surgery (MIVS). Methods Of 111,876 vitrectomies (70,585 20-G 41,291 MIVS) performed, 45 cases developed acute-onset, postoperative endophthalmitis. Results The rate of culture positive and culture negative endophthalmitis was 0.021% (2.1/10,000 surgeries) and 0.019% (1.9/10,000 surgeries) overall, 0.031% (3.1/10,000 surgeries) and 0.025% (2.5/10,000 surgeries) in 20G, and 0.005% (0.5/10,000 surgeries) and 0.007% (0.7/10,000 surgeries) in the MIVS group respectively. Potential predisposing factors were as follows: diabetes, 46.7%; vitrectomy for vascular retinopathies, 44.4%; and vitrectomy combined with anterior segment surgeries, 35.5%. The culture proven rates were 53.3% overall, 55.0% for 20G and 40.0% for MIVS. The most common organism was Pseudomonas aeruginosa for 20G. Klebsiella and Staphylococcus aureus were isolated in the two culture positive cases in MIVS group. The follow-up period for the patients with endophthalmitis was 586.14 ± 825.15 days. Seven were lost to follow up beyond one week. Of the remaining 38, 13 (34.2%) cases had a favorable visual outcome (i.e., best-corrected visual acuity [BCVA] > 5/200) and 24 (63.2%) had unfavorable visual outcome (BCVA < 5/200). Group with culture test results negative had significantly better outcomes (P < 0.05) as compared to those with positive. Conclusions MIVS does not increase the risk of endophthalmitis. Outcomes are poor despite appropriate treatment, particularly in cases with culture results positive.

MicroRNA Biomarkers in Neurodegenerative Diseases and Emerging Nano-Sensors Technology.

MicroRNAs (miRNAs) are essential small RNA molecules (20–24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Due to their roles in a variety of biological processes, the aberrant expression profiles of miRNAs have been identified as biomarkers for many diseases, such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases. In order to precisely, rapidly and economically monitor the expression of miRNAs, many cutting-edge nanotechnologies have been developed. One of the nanotechnologies, based on DNA encapsulated silver nanoclusters (DNA/AgNCs), has increasingly been adopted to create nanoscale bio-sensing systems due to its attractive optical properties, such as brightness, tuneable emission wavelengths and photostability. Using the DNA/AgNCs sensor methods, the presence of miRNAs can be detected simply by monitoring the fluorescence alteration of DNA/AgNCs sensors. We introduce these DNA/ AgNCs sensor methods and discuss their possible applications for detecting miRNA biomarkers in neurodegenerative diseases.

Cesium Toxicity Alters MicroRNA Processing and AGO1 Expressions in Arabidopsis thaliana

MicroRNAs (miRNAs) are short RNA fragments that play important roles in controlled gene silencing, thus regulating many biological processes in plants. Recent studies have indicated that plants modulate miRNAs to sustain their survival in response to a variety of environmental stimuli, such as biotic stresses, cold, drought, nutritional starvation, and toxic heavy metals. Cesium and radio-cesium contaminations have arisen as serious problems that both impede plant growth and enter the food chain through contaminated plants. Many studies have been performed to define plant responses against cesium intoxication. However, the complete profile of miRNAs in plants during cesium intoxication has not been established. Here we show the differential expression of the miRNAs that are mostly down-regulated during cesium intoxication. Furthermore, we found that cesium toxicity disrupts both the processing of pri-miRNAs and AGONOUTE 1 (AGO1)-mediated gene silencing. AGO 1 seems to be especially destabilized by cesium toxicity, possibly through a proteolytic regulatory pathway. Our study presents a comprehensive profile of cesium-responsive miRNAs, which is distinct from that of potassium, and suggests two possible mechanisms underlying the cesium toxicity on miRNA metabolism.