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Highly efficient nanoparticle-on-metallic-mirror (NPOM) systems with a large gap size exhibiting good plasmonic enhancement are desirable for numerous practical applications. Careful, explicit design optimization strategies are required for preparing NPOMs and it is especially important in utilizing spherical nanoparticles. In this work, a new design blueprint for evaluating the role of random facets in spherical nanoparticles was investigated in detail to realize optimal NPOMs. We found that a precise single facet positioned at the nanoparticle's cavity outperformed multiple random facets due to the gap mode contribution. Differences and changes in the plasmonic modes were interpreted with the help of three-dimensional surface charge density mappings. A high-performance, single, bottom-faceted NPOM device with a large gap size (example 20 nm) was realized having 80-50% facet design, resulting in excellent gap mode enhancement. We succeeded in fabricating single bottom-faceted NPOMs (the non-facet region had a smooth spherical surface) with a large-scale unidirectionality (2 cm × 1.5 cm). Simulations and experimental characterizations of these components displayed excellent agreement. Our highly efficient NPOM design with a large gap size(s) enables interesting practical applications in the field of quantum emitters, energy devices, fuel generation and plasmon chemistry.

Detecting trace amounts of explosives to ensure personal safety is important, and this is possible by using laser-based spectroscopy techniques. We performed surface-enhanced Raman scattering (SERS) using plasmonic nanogap substrates for the solution phase detection of some nitro-based compounds, taking advantage of the hot spot at the nanogap. An excitation wavelength of 785 nm with an incident power of as low as ≈0.1 mW was used to excite the nanogap substrates. Since both RDX and PETN cannot be dissolved in water, acetone was used as a solvent. TNT was dissolved in water as well as in hexane. The main SERS peaks of TNT, RDX, and PETN were clearly observed down to the order of picomolar concentration. The variations in SERS spectra observed from different explosives can be useful in distinguishing and identifying different nitro-based compounds. This result indicates that our nanogap substrates offer an effective approach for explosives identification.

A major limitation of RNA sequencing (RNA-seq) analysis of alternative splicing is its reliance on high sequencing coverage. We report DARTS (https://github.com/Xinglab/DARTS), a computational framework that integrates deep-learning-based predictions with empirical RNA-seq evidence to infer differential alternative splicing between biological samples. DARTS leverages public RNA-seq big data to provide a knowledge base of splicing regulation via deep learning, thereby helping researchers better characterize alternative splicing using RNA-seq datasets even with modest coverage.

The N6‐methyladenosine (m(6)A) RNA modification serves crucial functions in RNA metabolism; however, the molecular mechanisms underlying the regulation of m(6)A are not well understood. Here, we establish arginine methylation of METTL14, a component of the m(6)A methyltransferase complex, as a novel pathway that controls m(6)A deposition in mammalian cells. Specifically, protein arginine methyltransferase 1 (PRMT1) interacts with, and methylates the intrinsically disordered C terminus of METTL14, which promotes its interaction with RNA substrates, enhances its RNA methylation activity, and is crucial for its interaction with RNA polymerase II (RNAPII). Mouse embryonic stem cells (mESCs) expressing arginine methylation‐deficient METTL14 exhibit significantly reduced global m(6)A levels. Transcriptome‐wide m(6)A analysis identified 1,701 METTL14 arginine methylation‐dependent m(6)A sites located in 1,290 genes involved in various cellular processes, including stem cell maintenance and DNA repair. These arginine methylation‐dependent m(6)A sites are associated with enhanced translation of genes essential for the repair of DNA interstrand crosslinks; thus, METTL14 arginine methylation‐deficient mESCs are hypersensitive to DNA crosslinking agents. Collectively, these findings reveal important aspects of m(6)A regulation and new functions of arginine methylation in RNA metabolism.

Nearly all human multi-exonic genes undergo alternative splicing (AS) via regulation by RNA-binding proteins (RBPs), but few studies have examined the temporal dynamics of AS and its regulation during cell differentiation in the bone niche. We sought to evaluate how AS, under the control of RBPs, affects cell fate commitment during induced osteogenic differentiation of human bone marrow-derived multipotent stem/stromal progenitor cells (MSPCs). We generated a time-course RNA sequencing (RNA-seq) dataset representative of induced MSPC differentiation to osteoblasts. Our analysis revealed widespread AS changes, coordinated with differential RBP expression, at multiple time points, including many AS changes in non-differentially expressed genes. We also developed a computational approach to profile the dynamics and regulation of AS by RBPs using time-course RNA-seq data, by combining temporal patterns of exon skipping and RBP expression with RBP binding sites in the vicinity of regulated exons. In total we identified nine RBPs as potential key splicing regulators during MSPC osteogenic differentiation. Perturbation of one candidate,KHDRBS3, inhibited osteogenesis and bone formationin vitro, validating our computational prediction of “driver” RBPs. Overall, our work highlights a high degree of complexity in the splicing regulation of MSPC osteogenic differentiation. Our computational approach may be applied to other time-course data to explore dynamic AS changes and associated regulatory mechanisms in other biological processes or disease trajectories.

In realizing excellent plasmonic devices, a methodology based on flexibility and simplicity in fabrication, minimal sensitiveness to smaller nanoscale errors, larger dielectric layer thickness with superior device characteristics, and low-cost process is critically crucial for next-generation devices with multiple applications. One such attractive device is a plasmonic nanocavity, with numerous reports been already reported resulting in superior localized surface plasmon resonance (LSPR) and enhancement properties. The conventional spherical NP on a metallic mirror (NPOM) nanostructure’s plasmonic characteristics deteriorates with minor changes in dielectric layer thickness (t ≤ 6 nm). Alternatives like nanocube on mirror (NCOM), nanodisk on mirror (NDOM), provided better options towards LSPR tuning and near field enhancement. In recent times there are few reports based on faceted spherical NPOM design emerged. But however, the so far reported FNPOM nanostructures lacked the following: “facet width control, a clear SEM/TEM image of full geometry, and larger “t” with superior plasmonic characteristics”. In this work, for the first time, we report a clear FNPOM nanostructure with better control in facet fabrication using reactive thermal annealing (RTA) method. We used Ag NP on an Au mirror with SiO2 as a dielectric layer with different NP diameters of 50 nm, 70 nm and 100 nm with a precise facet width control (from 90% sphere to hemisphere). We employed a larger “t” ranging between 10 nm – 40 nm to display superior properties. From our dark field and LSPR mapping measurements, 70% of the sample are showed similar plasmonic characteristics from a 1 cm x 1 cm substrate. Our experiment results showed that it is possible to tune the LSPR resonance wavelength till 40 nm dielectric thickness reflecting it as a superior plasmonic nanocavity device. The reason behind this enhanced plasmonic characteristics is due to the introduction of facet in NPs and our three-dimensional finite difference time domain (3D FDTD) simulations results agreed well with experiment. For a final comparison, we checked our hemispherical shaped FNPOM versus NCOM design for NPs with diameter of 100 nm, where we find our FNPOM nanostructures showcased superior plasmonic properties.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

We report gap-dependent strong SERS enhancements and plasmonic couplings of hole/sphere nanostructures. The structure shows uniform sensitivity of less than 10% over the entire substrate, which comes from uniform narrow gaps over the substrate.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

Among myriads of distinct chemical modifications in RNAs, dynamic N6-methyladenosine (m(6)A) is one of the most prevalent modifications in eukaryotic mRNAs and non-coding RNAs. Similar to the critical role of chemical modifications in regulation of DNA and protein activities, RNA m(6)A modification is also observed to be involved in the regulation of diverse functions of RNAs including meiosis, fertility, development, cell reprogramming and circadian period. The RNA m(6)A modification is recognized by YTH domain containing family proteins comprising of YTHDC1-2 and YTHDF1-3. Here we focus on the nuclear m(6)A reader YTHDC1 and its regulatory role in alternative splicing and other RNA metabolic processes.

BACKGROUND: Uphill walking and downhill walking are the different forms of walking which causes cardiovascular changes in the body. By changing the gradient of the treadmill, we can change the severity of exercise by keeping the speed constant. AIM: This study aims to evaluate the cardiovascular changes during uphill, level, and downhill walking in young adults. STUDY DESIGN: This was an experimental study MATERIALS AND METHODS: Twenty participants (15 males and 5 females) having normal body mass index were made to walk on the treadmill at different gradients of −15%, 0%, and +15% at a preferential speed for a constant duration of 15 min. The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and rate pressure product (RPP) were recorded before and after walking. STATISTICAL ANALYSIS: Statistical analysis was done by ANOVA test using SPPSS, version 20 (IBM, Bangaluru, Karnataka ,India), where P RESULTS: There was a significant increase in HR (105.8 ± 20.06), SBP (133.3 ± 13.54), and RPP (14146 ± 3025) during uphill walking as compared to downhill and level walking. There were no significant changes in cardiovascular parameters when level walking and downhill walking were compared. DBP did not show any significant changes at any gradient of walking. CONCLUSION: Cardiovascular response increases during uphill walking, but level walking and downhill walking show no difference in cardiovascular response. These findings can be used to prescribe the exercises for young adults.